Induction of apoptosis in a non-small-cell human lung cancer cell line by isothiocyanates is associated with P53 and P21 but not with Bax.

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Induction of apoptosis in a non-small cell human lung cancer cell

line by isothiocyanates is associated with P53 and P21

Yu-Fang Kuang, Yue-Hwa Chen

*

School of Nutrition and Health Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan 110, ROC Received 8 January 2004; accepted 28 June 2004

Abstract

This study was aimed at examining the eﬀects of glucosinolate derivatives including phenylethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), and indole-3-carbinol (I3C), on the induction of apoptosis in human non-small cell lung carcinoma A549 cells. The results indicated that all tested compounds inhibited the growth of A549 cells in a concentration-dependent manner. Flow cytometric analyses and annexin V staining showed that induction of apoptosis occurred at low concentrations of PEITC and BITC (610 lM), and that necrosis occurred at higher concentrations of PEITC and BITC (25 lM); however, apoptosis was not the major pathway for the antiproliferative eﬀects of I3C. Furthermore, Western blot analyses demonstrated that increased expression of P53 and P21 proteins, but not Bax protein, were associated with PEITC- and BITC-induced apoptosis.

Keywords: Isothiocyanate; Indole-3-carbinol; Apoptosis; Lung adenocarcinoma A549 cells; P53; P21

1. Introduction

As the leading cause of cancer deaths in most devel-oped countries, lung cancer has garnered much atten-tion. Clinically, lung cancer is classiﬁed into two groups, small cell and non-small cell lung cancer. The latter is more prevalent, accounting for almost 80% of lung cancers. Non-small cell lung cancer is composed of several subtypes, including lung adenocarcinoma, which is the most common lung cancer in the US. Tra-ditionally, surgery, radiotherapy, and chemotherapy have been used to treat patients with lung cancer, but these treatments are usually accompanied by many side eﬀects. Therefore, it is important to develop new ap-proaches, such as apoptosis, for treating this type of lung cancer.

Apoptosis, or programmed cell death, is a common form of eukaryotic cell death. Apoptosis is a physiolog-ical cell suicide program that helps maintain homeosta-sis, in which cell death naturally occurs during tissue turnover (Samali et al., 1996; Staunton and Gaffney, 1998), but its aberrant activation and impairment may contribute to a number of diseases (Carson and Ribeiro, 1993;Thompson, 1995). For example, impaired apopto-sis may be a significant factor in the etiology of cancer, so it has been suggested that therapeutics that increase the regulation of apoptosis may provide a new opportu-nity for the treatment of cancer (Kerr et al., 1994; Staun-ton and Gaffney, 1998). Cells undergoing apoptosis usually show several cellular changes, including forma-tion of plasma membrane blebs, reducforma-tion in cell volume, chromatin condensation, and DNA fragmenta-tion, but cells retain their membrane and organelle integrity. Apoptosis is regulated by various gene prod-ucts including P53, Bcl, and Bax proteins (White, 1996;Staunton and Gaffney, 1998). In addition, several dietary phytochemicals that play significant roles in the

* _{Corresponding author. Tel.: +886 2 273 83464/61661x6555 118/;} fax: +886 2 273 73112.

E-mail address:[email protected](Y.-H. Chen).

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anticarcinogenic process have been identiﬁed to trigger the apoptotic death of cancer cells (Hibasami et al., 1996;Huang et al., 1998;Moragoda et al., 2001).

Cruciferous vegetables consisting of cabbage, Napa cabbage, broccoli, and cauliflower and their bioactive components, glucosinolates, have been reported to be associated with lowered risks of cancers (Graham and Mettlin, 1979; Stoewsand, 1995; Verhoeven et al., 1996;Craig, 1997). The degradation products of gluco-sinolates such as indole-3-carbinol (I3C) and isothiocya-nates, including b-phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC), have been shown to possess anticarcinogenic activities (Zhang and Talalay, 1994; Grubbs et al., 1995), and to induce apoptosis in various cancer cell lines (Huang et al., 1998;Ge et al., 1999; Bonnesen et al., 2001; Yang et al., 2002; Nachs-hon-Kedmi et al., 2003). PEITC has been shown to inhi-bit tobacco smoke-induced lung tumors in mice (Witschi et al., 1998), but related studies are not available on lung cancer cells. Therefore, the purpose of this study was to explore the effects of the cruciferous vegetable deriva-tives, PEITC, BITC and I3C, on the induction of apop-tosis in human non-small cell lung adenocarcinoma A549 cells. To understand the effects of these derivatives on cell growth, cell counts and cell proliferation were monitored. To identify the role of apoptosis in growth inhibitory effects, flow cytometric and annexin V stain-ing analyses were performed. Apoptosis-associated P53, P21, and Bax proteins were also examined. The re-sults obtained from this study may provide preclinical evidence on the potential use of compounds derived from cruciferous vegetables as cancer chemopreventive or chemotherapeutic agents.

2. Materials and methods

Chemicals and biochemicals. Indole-3-carbinol (I3C), b-phenylethyl isothiocyanate (PEITC), benzyl isothiocy-anate (BITC), dimethyl sulfoxide (DMSO), and propi-dium iodide (PI) were obtained from Sigma Chemical (St. Louis, MO). DulbeccoÕs modiﬁed Eagle medium (DMEM), fetal bovine serum, trypsin, trypan blue, and sodium bicarbonate were purchased from GIBCO BRL (Grand Island, NY). Absolute ethanol was from Merck (Darmstadt, Germany). All other laboratory chemicals were of the highest quality available and were purchased from Sigma Chemical and USB (Cleveland, OH).

Cell culture. The human lung adenocarcinoma cell line A549 was obtained from the Culture Collection and Research Center in Taiwan (CCRC 60074). Cells were grown as monolayers in DMEM supplemented with 10% fetal bovine serum at 37C in a 95% air, 5% CO2 atmosphere and routinely subcultured. PEITC

and BITC were dissolved in absolute ethanol, and I3C

was dissolved in DMSO; the concentrations of absolute ethanol and DMSO added to the media never exceeded 0.5% (v/v) and 0.2% (v/v), respectively.

Cell growth and cell proliferation assays. To evaluate the eﬀects of PEITC, BITC, and I3C on the growth of cells, A549 cells (5· 104

/well) were plated in 6-well plates and cultivated in the presence of test compounds. Viable cells were estimated by the trypan blue dye exclu-sion method, and numbers of cells were counted with a Coulter counter.

Cell proliferation was examined with a CellTiter 96 Aqueous One Solution Cell Proliferation Assay@ kit (Promega, Madison, WI). Basically, 103cells/well were plated in 96-well plates, and were treated with test com-pounds for 3 days. The rate of proliferation was then determined by converting a tetrazolium compound, 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), to a colored formazan product by live cells as detected by a micro-plate reader at OD492 nm.

Flow cytometric analysis. To analyze the distribution of cellular DNA content, flow cytometric analysis was performed. After being treated with the test compounds for 24 h, cells were collected, suspended, and fixed with 75% ethanol on ice. Fixed cells were incubated with 3 lg/ml DNAase-free RNAase A in PBS for 30 min at 37C. After staining with PI (20 lg/ml), cells were ana-lyzed by FACScan laser flow cytometry (Becton Dicken-son, San Jose, CA) with excitation at 488 nm.

Annexin V staining assay. In the beginning of apopto-sis, phosphatidylserine inside of the cell membrane is inverted to the outside. Therefore, binding of phosphat-idyl serine to annexin V (green) and DNA to PI (red) can be used for the detecting apoptosis and its various stages. Annexin V staining was carried out with annexin V-FITC Apoptosis Detection kits (Oncogene, Boston, MA) following the manufacturerÕs instructions. In brief, media binding reagent and annexin V-fluorescein isoth-iocyanate were added to the cells for 15 min in the dark. Cells were treated with binding buffer and PI, and were then photographed under a fluorescent microscope.

SDS–PAGE and Western blot analyses. To determine whether P53, Bax, and P21 are associated with the induction of apoptosis, Western blot analysis was per-formed. Cell lysate was isolated by treating cells with lysis solution (150 mM NaCl, 1% Triton X-100, 10 mM Tris, pH7.4, 5 mM EDTA, and 1 mM phenyl-methyl-sulfonylﬂuoride), and the protein content was determined using a Bio-rad protein assay kit. After sep-aration by 12.5% sodium dodecyl sulfate (SDS)–poly-acrylamide gel electrophoresis (SDS–PAGE), the proteins were electroblotted onto a nitrocellulose mem-brane, and the blots were incubated with monoclonal antimouse P53 (DO-1), polyclonal Bax (N-20) (Santa Cruz Biotechnology, Santa Cruz, CA), or mouse mono-clonal P21WAF1/CIP1antibodies (BD Transduction Lab,

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Los Angeles, CA). Finally, the blot was treated with per-oxidase-conjugated IgG (Santa Cruz Biotechnology), and speciﬁc bindings of these antibodies were detected using an enhanced chemiluminescent ECL Western detection system (Amersham Biosciences, Buckingham-shire, England).

Statistical analysis. Data are expressed as the mean ± standard deviation (SD). One-way ANOVA fol-lowed by FisherÕs test was used to determine the statisti-cal differences among groups using SAS software version 6.12 (SAS Institute, Cary, NC). The significance of mean differences was based on a p value of <0.05.

3. Results

To examine the eﬀects of cruciferous vegetable deriv-atives on the growth of A549 cells, cells were treated with various concentrations of PEITC, BITC, and I3C, and numbers of cells were determined after 3 and 5 days. Fig. 1 indicates that all three compounds

inhibited the growth of A549 cells in concentration-dependent manners. PEITC and BITC, at the highest concentration (25 lM), completely suppressed the growth of cells. However, I3C, at 250 lM, produced 67% growth inhibition after 5 days of treatment. Results obtained from the MTS assay illustrate comparable dose–response patterns (Fig. 2), and the approximate IC50 values for PEITC, BITC, and I3C were 7.5, 3,

and 200 lM, respectively.

To explore the mechanisms of PEITC, BITC, and I3C on the inhibition of cell growth, the DNA content of treated cells was determined by ﬂow cytometric anal-ysis.Fig. 3indicates that the distribution of DNA con-tents in PEITC-treated cells diﬀered from that of control cells after 24 h treatment. PEITC-treated cells showed a concentration-dependent increase in the number of cells with subdiploid DNA contents (sub G1), i.e., apoptotic

cells, which reached a maximum at 10 lM, and this change was accompanied by a decreased G0/G1 peak

and an increased G2/M peak. BITC produced similar

results to those of PEITC (data not shown). By contrast,

0 0.3 0.6 0.9 1.2 1.5 1.8 1 3 5 Time (d) Cell number (×10 6 ) control 2.5 µM 5 µM 7.5 µM 10 µM 25 µM

*

0 0.3 0.6 0.9 1.2 1.5 1.8 1 3 5 Time (d) Cell number (×10 6 ) control 2.5 µM 5 µM 7.5 µM 10 µM 25 µM

*

**

*

0 0.4 0.8 1.2 1.6 1 3 5 Time (d) Cell number (×10 6 ) control 50 µM 100 µM 150 µM 200 µM 250 µM

*

(A) (B) (C)

Fig. 1. Eﬀects of PEITC, BITC, and I3C on the growth of A549 cells. Cells were treated with various concentrations of PEITC (A), BITC (B) and I3C (C), and then were collected and counted with a Coulter counter at the times indicated. Values represent the mean ± SD from three measurements. *p < 0.05 compared with the control at the same time point.

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I3C (250 lM) did not aﬀect the distribution of DNA content after 24- or 48-h treatments. A slight increase in the proportion of sub G1cells (from 0.6% to 1.3%)

and an increased G0/G1 peak (from 79% to 90%) were

observed in cells treated with I3C for 72 h (data not shown).

As a cell undergoes apoptosis, phosphatidylserine (PS) is translocated from the inner to the outer mem-brane of the cell. Because annexin V binds to PS with high aﬃnity, the apoptotic cells can be observed with ﬂuorescent microscopy.Fig. 4shows that 10 lM PEITC increased the numbers of annexin V-positive (green- or orange-colored) cells, indicating that the cells had undergone apoptosis. However, more PI-positive (red color) cells were present after treatment with 25 lM PEITC, suggesting that late apoptotic or necrotic cells

are present at high concentrations of PEITC (Fig. 4C). Similar results were also observed in BITC-treated cells (data not shown). These results together with the results obtained from ﬂow cytometry suggested that PEITC and BITC induce apoptosis in A549 cells at low concen-trations (610 lM), and necrosis at a higher concentra-tion (25 lM).

A variety of proteins have been reported to be asso-ciated with the apoptosis pathway, so the effects of PEITC and BITC on various apoptosis-associated pro-teins were examined. Western blot analyses (Fig. 5) showed that PEITC enhanced the expression of P53 and P21WAF1/CIP1 proteins in concentration dependent manners, and respectively produced 160% and 270% in-creases compared to that of the control at 10 lM. Although BITC also enhanced the expressions of P53 and P21WAF1/CIP1proteins, they were not concentration dependent, and showed a lesser effect on P21WAF1/CIP1 expression compared to did PEITC. On the contrary, neither PEITC nor BITC affected the expression of cyto-solic Bax protein.

4. Discussion

Diﬀerent epidemiological studies have indicated that diet and cancers are closely associated (Cummings and Bingham, 1998; Labadarios and Parke, 1999). People who consume higher amount of fruits and vegetables have a lower risk of various types of cancers. Various studies demonstrate that fruits and vegetables contain natural occurring compounds that possess anticarcino-genic properties (Steinmetz and Potter, 1996; Craig,

0 20 40 60 80 100 1 10 100 1000 Concentration (µM) Cell viability (%) PEITC BITC I3C

Fig. 2. Eﬀects of PEITC, BITC, and I3C on the viability of A549 cells. Cells were treated with various concentrations of PEITC, BITC, and I3C for 72 h. Cell viability was then determined by an MTS assay kit. Values represent the mean ± SD from four measurements.

Fig. 3. Representative histograms of cytometric analysis after treatment of cells with PEITC. A549 cells were treated with various concentrations of PEITC for 24 h. Cells were then collected, ﬁxed and stained with PI in the ﬂow cytometric analysis for DNA content. Values represent the mean from four measurements.

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1997), such as glucosinolates in cruciferous vegetables (Stoewsand, 1995). Isothiocyanates and indoles are two major groups of autolytic breakdown products of glucosinolates, and both exhibit protective activities against cancers (Zhang and Talalay, 1994; McDanell et al., 1988). PEITC and I3C have been shown to mod-ulate the activity of xenobiotic-metabolizing enzymes

(Bradﬁeld and Bjeldanes, 1984;Guo et al., 1992), to sup-press lipopolysaccharide-induced nitric oxide produc-tion (Chen et al., 2003), and to induce apoptosis in cancer cells (Huang et al., 1998;Ge et al., 1999; Bonne-sen et al., 2001;Chinni et al., 2001), pathways that have been suggested to be involved in the anticarcinogenic process. In the present study, for the ﬁrst time, we have demonstrated that PEITC, BITC, and I3C, inhibit the growth of lung adenocarcinoma A549 cells. The isothio-cyanate derivatives, PEITC and BITC, show higher po-tency in inhibiting proliferation by acting through induction of apoptosis at low concentrations (<10 lM)

Fig. 4. Fluorescent microscopy of PEITC-induced morphological changes in A549 cells. Cells were treated with ethanol (A), 10 lM PEITC (B), or 25 lM PEITC (C) for 24 h. Cells were then stained with annexin V-FITC and PI and photographed under a ﬂuorescence microscope. Green color indicates apoptotic cells at an early stage, green and red (orange) indicate cells at the middle or late stage of apoptosis, and red indicates necrotic cells. (For interpretation of the references in color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 5. Eﬀects of various concentrations of PEITC or BITC on the expressions of P53, P21, and Bax proteins in A549 cells. (A) Cells were treated with various concentrations of PEITC or BITC, and the cytosolic protein was extracted after 24 h. 70 lg of cytosolic protein was separated by SDS–PAGE, and P53, P21, and Bax proteins were respectively detected. This experiment was repeated three times, and similar results were obtained. (B) Densitometric quantiﬁcation of the proteins.

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and through necrosis at a high concentration (25 lM), whereas the indolic derivative I3C, with higher IC50

value (200 lM), by acting through an apoptosis-inde-pendent pathway. Different dietary phytochemicals that play significant roles in the anticarcinogenic process have been identified to trigger the apoptotic death of human lung cancer cells, including curcumin (Pillai et al., 2004), kaempferol (Nguyen et al., 2003), and tea polyphenols (Suganuma et al., 1999), with IC50 values

greater than 22 lM. Therefore, the results obtained from this study introduce new candidates of treatment for non-small cell lung cancers and provide the molecu-lar mechanisms of PEITC and BITC in the induction of apoptosis in lung adenocarcinoma cells.

The antiproliferative eﬀect of I3C in A549 cells was independent of the induction of apoptosis, although I3C, at concentrations of 50–250 lM, induces apoptosis in hormone-related cell lines including human breast cancer MCF-7 and human prostate cancer LNCaP cells (Ge et al., 1999;Nachshon-Kedmi et al., 2003). In addi-tion, I3C shows observable toxicity to the human colon cell lines, LS-174 and Caco-2, only at high concentra-tions with IC50 values of 600 lM (Bonnesen et al.,

2001); this suggests that hormone-responsive cells are more sensitive to I3C treatment. Because I3C is an estrogen receptor antagonist (Meng et al., 2000), and I3C-induced apoptosis in MCF-7 cells has been sug-gested to be estrogen receptor related (Ge et al., 1999), it is conceivable to explain that apoptosis in A549 cells not being induced by 250 lM I3C is due to its cell-type-dependence eﬀect. On the other hand, mecha-nisms other than apoptosis may be involved in the growth inhibitory eﬀect of I3C.Chinni et al. (2001) sug-gested that I3C induces G1 cell cycle arrest in poorly

diﬀerentiated prostate cancer PC-3 cells. Whether I3C inhibits the growth of A549 cells through induc-tion of G1 cell cycle arrest needs to be investigated

further.

Both apoptosis and necrosis were associated with the antiproliferative eﬀects of PEITC and BITC in A549 cells, in which apoptosis was mainly induced by low con-centrations (610 lM), whereas necrosis was essentially associated with higher concentrations (25 lM) of PEITC and BITC. These results are consistent with sev-eral other studies showing that PEITC and BITC (<20 lM) induce apoptosis in other cell lines, including human prostate cancer cells, human colon cancer cells, human HeLa cells, human leukemia Jurkat T-cells, mouse epidermal cells, and rat liver epithelial cells (Chen et al., 1998;Huang et al., 1998;Yu et al., 1998; Bonne-sen et al., 2001; Nakamura et al., 2002). Meanwhile, some of these studies also showed that PEITC and/or BITC at concentrations greater than 30 or 50 lM induce necrosis in human HeLa cells, human leukemia Jurkat T-cells, and rat liver epithelial RL34 cells (Chen et al., 1998;Yu et al., 1998;Nakamura et al., 2002). Therefore,

PEITC and BITC at concentrations below 10 lM are able to induce apoptosis in various cancer cell lines.

Several pieces of evidence have indicated that signals leading to activation of a variety of gene products, such as P53, P21 and Bcl-2 family proteins consisting of Bax, are important in the regulation and execution of apopto-sis induced by various stimuli (Kerr et al., 1994). The induction of apoptosis by PEITC and BITC is associ-ated with the increased expression of P53 suggesting the pivotal roles of P53 in this process, and this is con-sistent with the study byHuang et al. (1998)who dem-onstrated that apoptosis induced by PEITC occurs through a P53-dependent pathway in mouse epidermal C1 41 cells. P53 is a tumor suppressor protein and tran-scription factor that plays a substantial role in apopto-sis. The increased expression of P53 may act through certain pathways, including mitochondrial cytochrome c release and Fas/APO1 signaling, to enhance apopto-sis-downstream caspase enzymes to induce apoptosis (el Deiry, 1998; Shen and White, 2001). PEITC and BITC have been shown to upregulate caspase-3 and thus induce apoptosis in HeLa cells (Yu et al., 1998), so the increased P53 may act through increasing activities of caspases to induce apoptosis in A549 cells. P53 also plays an important role in cell cycle regulation. Studies have shown that P53 mediates apoptosis by a mecha-nism independent of that of cell cycle arrest (el Deiry, 1998;Shen and White, 2001). P21, a universal inhibitor of cyclin-dependent kinases (CDKs) (Xiong et al., 1993), is a P53-regulated protein. Upregulation of P21WAF1/CIP1 by diﬀerent chemopreventive agents is associated with G2/M phase arrest in the cell cycle (Lian

et al., 1998; Bilim et al., 2000). Because PEITC- and BITC-induced apoptosis in A549 cells is associated with increased expressions of P53 and P21WAF1/CIP1, the mechanism of cell cycle arrest on the cell death signaling of PEITC and BITC cannot be ruled out. On the other hand,Xiao and Singh (2002)showed that P53 is not re-quired for PEITC-induced apoptosis in human prostate cancer PC-3 cells. Because the expression of P21 can also be regulated by other pathways that are independ-ent of P53 (Zeng and el Deiry, 1996;el Deiry, 1998), the increased expression of P21 might not be directly related to the increased expression of P53.

Functions of P53 are mainly regulated by phosphory-lation. Diﬀerent kinases including mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK1) have been suggested to be involved in the phos-phorylation of P53 (Milczarek et al., 1997). Several pieces of evidence have demonstrated that PEITC and BITC induce apoptosis in various cell types through activation of JNK (Yu et al., 1996; Chen et al., 1998) or through suppression of JNK phosphatase activity (Chen et al., 2002). It is reasonable to postulate that the increased expression of P53 by PEITC and BITC may be mediated by the activation of JNK, and that

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the increased P53 thus leads to increases in the activities of caspases and/or the expression of P21 to induce apop-tosis or to cause cell cycle arrest. Therefore, treatment with PEITC and BITC may induce cell death in lung carcinoma cells, ultimately leading to the prevention of cancer.

Bax, another P53-regulated protein, is a member of the Bcl-2 family, and the ratio of Bax to Bcl-2 has been suggested to be a determinant of whether cells live or die (el Deiry, 1998;Shen and White, 2001), i.e., the Bax–Bax homodimer acts as an inducer of apoptosis, whereas the Bax–Bcl2 heterodimer acts as an inhibitor of apoptosis. Therefore, the existence of Bcl-2 and the balance be-tween Bax and Bcl-2 may direct the process of apopto-sis. Additionally, Bax dimers appear to control apoptosis at the level of mitochondrial cytochrome c re-lease (el Deiry, 1998), so apoptosis induced by PEITC and BITC in A549 cells might not be related to the mit-ochondrial cytochrome c pathway. On the other hand, because the Bax protein undergoes subcellular redistri-bution during apoptosis (Hsu et al., 1997; Gross et al., 1998), the total amounts of Bax protein as shown in this study may not be enough to disclaim the independence of Bax in this process. Therefore, further experiments are required to conﬁrm the role of Bax on the PEITC-and BITC-induced apoptosis in A549 cells.

In conclusion, we have demonstrated that the crucif-erous vegetable isothiocyanate derivatives, PEITC and BITC, are able to inhibit the growth of A549 cells by inducing apoptosis at low concentrations and necrosis at high concentrations. The induction of apoptosis is associated with increased expression of P53 and P21 proteins. On the other hand, I3C, an indolic derivative of cruciferous vegetables, also possesses an antiprolifer-ative eﬀect in A549 cells, but not through induction of apoptosis. However, further experiments focusing on molecular mechanisms are needed to establish the role of the cruciferous derivatives, PEITC, BITC, and I3C, as chemopreventive or therapeutic agents against lung cancer.

Acknowledgment

This work was supported by a grant from Taipei Medical University (TMU91-Y05-A125), Taiwan, ROC.

References

Bilim, V., Kawasaki, T., Takahashi, K., Tomita, Y., 2000. Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21WAF1/CIP1genes. Journal of Experimental & Clinical Cancer Research 19, 483–488.

Bonnesen, C., Eggleston, I.M., Hayes, J.D., 2001. Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can

both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Research 61, 6120–6130. Bradﬁeld, C.A., Bjeldanes, L.F., 1984. Eﬀect of dietary indole-3-carbinol on intestinal and hepatic monooxygenase, glutathione S-transferase and epoxide hydrolase activities in the rat. Food and Chemical Toxicology 22, 977–982.

Carson, D.A., Ribeiro, J.M., 1993. Apoptosis and disease. The Lancet 341, 1251–1254.

Chen, Y.H., Dai, H.-J., Chang, H.-P., 2003. Suppression of inducible nitric oxide production by indole and isothiocyanate derivatives from Brassica plants in stimulated macrophages. Planta Medica 69, 696–700.

Chen, Y.-R., Han, J., Kori, R., Kong, A.-N.T., Tan, T.-H., 2002. Phenylethyl isothiocyanate induces apoptotic signaling via sup-pressing phosphatase activity against c-Jun N-terminal kinase. Journal of Biological Chemistry 277, 39334–39342.

Chen, Y.-R., Wang, W., Kong, A.-N.T., Tan, T.-H., 1998. Molecular mechanisms of c-Jun N-terminal kinase-mediated apoptosis induced by anticarcinogenic isothiocyanates. The Journal of Biological Chemistry 273, 1769–1775.

Chinni, S.R., Li, Y., Upadhyay, S., Koppolu, P.K., Sarkar, F.H., 2001. Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene 20, 2927–2936.

Craig, W.J., 1997. Phytochemicals: guardians of our health. Journal of the American Dietetic Association 97 (Suppl. 2), S199–S204. Cummings, J.H., Bingham, S.A., 1998. Fortnightly review: diet and

the prevention of cancer. British Medical Journal 317, 1636–1640. el Deiry, W.S., 1998. Regulation of p53 downstream genes. Seminars

in Cancer Biology 8, 345–357.

Ge, X., Fares, F.A., Yannai, S., 1999. Induction of apoptosis in MCF-7 cells by indole-3-carbinol is independent of p53 and Bax. Anticancer Research 19, 3199–3204.

Graham, S., Mettlin, C., 1979. Diet and colon cancer. American Journal of Epidemiology 109, 1–20.

Gross, A., Jockel, J., Wei, M.C., Korsmeyer, S.J., 1998. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. The EMBO Journal 17, 3878–3885. Grubbs, C.J., Steele, V.E., Casebolt, T., Juliana, M.M., Eto, I.,

Whitaker, L.M., Dragnev, K.H., Kelloﬀ, G.J., Lubet, R.L., 1995. Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol. Anticancer Research 15, 709–716.

Guo, Z., Smith, T.J., Wang, E., Sadrieh, N., Ma, Q., Thomas, P.E., Yang, C.S., 1992. Eﬀects of phenethyl isothiocyanate, a carcino-genesis inhibitor, on xenobiotic-metabolizing enzymes and nitro-samine metabolism in rats. Carcinogenesis 13, 2205–2210. Hibasami, H., Achiwa, Y., Fujukawa, T., Komiya, T., 1996. Induction

of programmed cell death (apoptosis) in human lymphoid leuke-mia cells by catechin compounds. Anticancer Research 16, 1943– 1946.

Hsu, Y.-T., Wolter, K.G., Youle, R.J., 1997. Cytosol-to-membrane redistribution of Bax and Bcl–XLduring apoptosis. Proceedings of National Academy Sciences, USA 94, 3668–3672.

Huang, C., Ma, W.-Y., Li, J., Hecht, S.S., Dong, Z., 1998. Essential role of p53 in phenethyl isothiocyanate-induced apoptosis. Cancer Research 58, 4102–4106.

Kerr, J.F.R., Winterford, C.M., Harmon, B.V., 1994. Apoptosis: its signiﬁcance in cancer and cancer therapy. Cancer 73, 2013–2026. Labadarios, D., Parke, D.V., 1999. Nutrition and diet in the

prevention and treatment of cancer. Journal of Clinical and Biochemical Nutrition 26, 135–153.

Lian, F., Bhuiyan, M., Li, Y.-W., Wall, N., Kraut, M., Sarkar, F.H., 1998. Genistein-induced G2-M arrest, p21

WAF1

upregulation, and apoptosis in a non-small-cell lung cancer cell line. Nutrition and Cancer 31, 184–191.

McDanell, R., McLean, A.E.M., Hanley, A.B., Heaney, R.K., Fenwick, G.R., 1988. Chemical and biological properties of indole

(8)

glucosinolates (glucobrassicins): a review. Food and Chemical Toxicology 26, 59–70.

Meng, Q., Yuan, F., Goldberg, I.D., Rosen, E.M., Auborn, K., Fan, S., 2000. Indole-3-carbinol is a negative regulator of estrogen receptor-a signaling in human tumor cells. Journal of Nutrition 130, 2927–2931.

Milczarek, G.J., Martinez, J., Bowden, G.T., 1997. P53 phosphory-lation: biochemical and functional consequences. Life Sciences 60, 1–11.

Moragoda, L., Jaszewski, R., Majumdar, A.P.N., 2001. Curcumin induced modulation of cell cycle and apoptosis in gastric and colon cancer cells. Anticancer Research 21, 873–878.

Nachshon-Kedmi, M., Yannai, S., Haj, A., Fares, F.A., 2003. Indole-3-carbinol and 3,30_{-diindolylmethane induce apoptosis in human}

prostate cancer cells. Food and Chemical Toxicology 41, 745–752. Nakamura, Y., Kawakami, M., Yoshihiro, A., Miyoshi, N., Ohigash, K., Kawai, K., Osawa, T., Uchida, K., 2002. Involvement of the mitochondrial death pathway in chemopreventive benzyl isothio-cyanate-induced apoptosis. Journal of Biological Chemistry 277, 8492–8499.

Nguyen, T.T.T., Tran, E., Ong, C.K., Lee, S.K., Do, P.T., Huynh, T.H., Nguyen, T.H., Lee, J.J., Tan, Y., Ong, C.S., Huynh, H., 2003. Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK–MAPK. Journal of Cellular Physiology 197, 110–121.

Pillai, G.R., Srivastava, A.S., Hassanein, T.I., Chauhan, D.P., Carrier, E., 2004. Induction of apoptosis in human lung cancer cells by curcumin. Cancer Letters 208, 163–170.

Samali, A., Gorman, A.M., Cotter, T.G., 1996. Apoptosis: the story so far . . . Experientia 52, 933–941.

Shen, Y., White, E., 2001. p53-Dependent apoptosis pathways. Advances in Cancer Research 82, 55–84.

Staunton, M.J., Gaﬀney, E.F., 1998. Apoptosis: basic concepts and potential signiﬁcance in human cancer. Archives of Pathology and Laboratory Medicine 122, 310–319.

Steinmetz, K.A., Potter, J.D., 1996. Vegetables, fruit, and cancer prevention: a review. Journal of the American Dietetic Association 96, 1027–1039.

Stoewsand, G.S., 1995. Bioactive organosulfur phytochemicals in Brassica oleracea vegetables––a review. Food and Chemical Toxi-cology 33, 537–543.

Suganuma, M., Okabe, S., Kai, Y., Sueoka, N., Sueoka, E., Fujiki, H., 1999. Synergistic eﬀects of epigallocatechin gallate with ()-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9. Cancer Research 59, 44–47. Thompson, C.B., 1995. Apoptosis in the pathogenesis and treatment of

disease. Science 267, 1456–1462.

Verhoeven, D.T.H., Goldbohm, R.A., van Poppel, G., Verhagen, H., van den Brandt, P.A., 1996. Epidemiological studies on Brassica vegetables and cancer risk. Cancer Epidemiology, Biomarkers & Prevention 5, 733–748.

White, E., 1996. Life, death, and the pursuit of apoptosis. Genes and Development 10, 1–15.

Witschi, H., Espiritu, I., Yu, M., Willits, N.H., 1998. The eﬀects of phehethyl isothiocyanate, N-acetylcysteine and green tea on tobacco smoke-induced lung tumors in strain A/J mice. Carcino-genesis 19, 1789–1794.

Xiao, D., Singh, S.V., 2002. Phenethyl isothiocyanate-induced apop-tosis in p53-deﬁcient PC-3 human prostate cancer cell line is mediated by extracellular signal-regulated kinases. Cancer Research 62, 3615–3619.

Xiong, Y., Hannon, G.J., Zhang, H., Beach, D., 1993. P21 is a universal inhibitor of cyclin kinases. Nature 366, 701–704. Yang, Y.-M., Conaway, C.C., Chiao, J.W., Wang, C.-X., Amin, S.,

Whysner, J., Dai, W., Reinhardt, J., Chung, F.-L., 2002. Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and isothiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis. Cancer Research 62, 2–7.

Yu, R., Jiao, J.J., Duh, J.L., Tan, T.H., Kong, A.N., 1996. Phenethyl isothiocyanate, a natural chemopreventive agent, activates c-Jun N-terminal kinase 1. Cancer Research 56, 2954–2959.

Yu, R., Mandlekar, S., Harvey, K.J., Ucker, D.S., Kong, A.-N.T., 1998. Chemopreventive isothiocyanates induce apoptosis and caspase-3-like protease activity. Cancer Research 58, 402–408. Zeng, Y.X., el Deiry, W.S., 1996. Regulation of p21WAF1/CIP1

expression by p53-independent pathways. Oncogene 12, 1557– 1564.

Zhang, Y., Talalay, P., 1994. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Research 54 (Suppl.), 1976S–1981S.