P53-Independent Apoptosis Induced by Genistein in Lung Cancer Cells

Abstract: Lung cancer is the leading cause of cancer-related deaths in the world, with increasing incidence in many developed countries. Epidemiological data suggest that consumption of soy products may be associated with a decreased risk of cancer. Despite the association of nutrition and cancer, the molecular mechanisms by which the active metabolite in the soy diet, genistein, exerts its biological response have not been studied. We previously showed that genistein can inhibit the growth of H460 non-small-cell lung cancer (NSCLC) cells in vitro. To explore the molecular mechanisms by which genistein inhibits the growth of NSCLC cells, we investigated cell growth inhibition, modulation in gene expression, and induction of apoptosis by genistein in H460 cells, which harbor wild-type p53, and H322 cells, which possess mutated p53. Genistein was found to inhibit H460 and H322 cell growth in a dose-dependent manner. Staining with 4,6-diamidino-2-phenylindole, poly(ADP-ribose) polymerase cleavage, and flow cytometric apoptosis analysis were used to investigate apoptotic cell death, and the results show that 30 muM genistein causes cell death via a typical apoptotic pathway. Western blot analysis demonstrated upregulations of p21WAF1 and Bar by genistein in wild-type and mutant p53 cell lines. Furthermore, cells treated with genistein showed an increased expression of endogenous wild-type p53, while the level of the mutant p53 protein remained unchanged. From these results, we conclude that genistein induces apoptosis in NSCLC cells through a p53-independent pathway and, thus, may act as an anticancer agent.

Introduction
Lung cancer is the leading cause of cancer-related death ( 1). The high prevalence, high death rate, and ineffective therapy have spurred the search for novel strategies in the prevention of lung cancer. Smoking cessation is the most effective prevention tool; however, because smoking cessation programs in the teenage population have met with limited success, chemoprevention is gaining more attention. This innovative approach aims to decrease overall cancer morbidity and mortality by using substances that are capable of preventing cancer development or progression. Several classes of compounds have been evaluated by in vitro assays for this purpose. One of them, genistein, is very effective in gastrointestinal, prostate, breast, and hematologic cancer cells ( 2-4). Genistein ( 5, 7, 4'-trihydroxyisoflavone), an isoflavonoid derived from soybeans, is a potent inhibitor of protein tyrosine kinase (PTK) ( 5-7). PTK is known to play a key role in growth factor-related signal modulation and in programmed cell death, known as apoptosis ( 8-11). Numerous in vitro studies have shown that genistein can inhibit cell proliferation and cause cell cycle arrest. In addition, recent studies have shown that genistein can induce apoptosis ( 2, 11).

Apoptosis is an active, energy-dependent process of cellular self-destruction. The morphological changes include condensation of nuclear contents into clumps of heterochromation adjacent to the nucleus, nuclear fragmentation, and final packaging of the nuclear fragments into membrane-enclosed apoptotic bodies. Apoptosis can be triggered by various external stimuli, including DNA-damaging agents, such as chemotherapeutic drugs and irradiation ( 12). p53, a tumor suppressor and transcription factor, is a critical regulator of the cellular response to DNA damage ( 13). Because p53 functions as a tumor suppressor, mutations of the p53 gene are common in cancers, p21WAF1 is a downstream effector of p53 and has been suggested to mediate p53-induced growth arrest that is triggered by DNA damage. It has, however, also been shown that expression of p21WAF1can be induced through a p53-independent pathway ( 14). p21WAF1 has been shown to induce apoptosis in certain cell types ( 15, 16); however, its direct role in apoptosis is unclear. Recently, the existence of a Bcl-2-related protein family, including Bax, has been reported ( 17, 18). It was shown that the proteins of the Bcl-2 family interact to form various homo- and heterodimers that have been suggested to stimulate or inhibit apoptotic cell death ( 19, 20). Knudson and Korsmeyer ( 21) showed that Bcl-2 and Bax function independently to regulate apoptotic cell death in vivo. A single copy of Bax can promote apoptosis in the absence of Bcl-2,whereas Bcl-2 represses apoptosis in the absence of Bax ( 21). Many studies on apoptotic regulation have been performed; however, the mechanism of apoptosis is unclear. The purpose of our study was to investigate the molecular mechanisms by which genistein exerts its effects on lung cancer cells and to provide the scientific rationale for using genistein as a chemopreventive or therapeutic agent against non-small-cell lung cancer (NSCLC).

Materials and Methods
Cell Culture and Growth-Inhibition Studies
The human lung cancer cell lines H460 and H322, obtained from the M. D, Anderson Cancer Center (University of Texas at Houston), were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine in a 5% CO2 atmosphere at 37degreesC. Cells (2.4 x 105) were plated in six-well dishes and incubated overnight. The cells were then treated with 0, 5, 10, 20, 30, and 50 muM genistein over three days. Sodium carbonate (0.1 M) was used for dissolving genistein to prepare 10 mM stock. This stock solution was used for experiments. The control culture used in our studies received appropriate medium with sodium carbonate concentration equivalent to the highest concentration of genistein. After one, two, and three days of treatment with genistein, the cells were trypsinized and counted using a hemocytometer.

Labeling With 4,6-Diamidino-2-Phenylindole
Control cells and cells treated with increasing doses of genistein (30-50muM) were plated on coverslips, harvested at one to three days posttreatment, and fixed in ethanol. The cells were then incubated with 4,6-diamidino-2-phenylin-dole (DAPI) at 1 mg/ml methanol for 15 minutes at 37degreesC. The staining solution was removed. Coverslips were washed with methanol, mounted on slides, and visualized under a fluorescence microscope.

Flow Cytometric Analysis for Apoptosis
Staining with 7-amino actinomycin D ( 7-AAD) and flow cytometry were used to detect apoptotic cells ( 22). Cells were seeded on 100-mm dishes, then treated with or without 50 muM genistein for one, two, or three days. Cells were harvested, centrifuged, and resuspended in phosphate-buffered saline (PBS). A total of 100 mul of 7-AAD solution (200 mug/ml) was added to 106 cells that were then suspended in PBS and mixed thoroughly. Cells were stained for 20 minutes at 4degreesC while protected from light and then pelleted by centrifugation. The cell pellets were resuspended in 500 mul of 2% paraformaldehyde solution (Sigma Chemical, St. Louis, MO). Unstained fixed cells were used as a negative control. Samples were analyzed on a FACscan (Becton Dickinson, Sunnyvale, CA) with 30 minutes of fixation. Data were processed by Lysys II software (Becton Dickinson). Scattergrams were generated by combining forward light scatter with 7-AAD fluorescence, and regions were drawn around clear-cut populations having negative, dim, and bright fluorescence.

Poly(ADP-Ribose) Polymerase Assay
Control cells and cells treated with 50 muM genistein for two days were washed twice with icecold PBS, harvested by scraper, and centrifuged. The cell pellets were lysed in lysis buffer [10 mM tris(hydroxymethyl)aminomethane (Tris).HCl (pH 7.1), 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 muM sodium orthovanadate, 2 mM iodoacetic acid, 5 muM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100]. The lysates were kept on ice for 30 minutes and vigorously vortexed before centrifugation at 12,500 g for 20 minutes. Fifty micrograms of total protein were resolved on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the membrane. The membrane was incubated with primary monoclonal antihuman poly(ADP-ribose) polymerase (PARP) antibody (1:5,000; Biomol), washed with Trisbuffered saline-Tween 20, and incubated with secondary antibody conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Pierce, Rockford, IL).

Protein Extraction and Western Blot Analysis for p21WAF1
H460 and H322 cells were plated on 100-mm dishes and treated with 30 and 50 muM genistein or sodium carbonate, as described previously. The cells were removed carefully from the cell culture dish with use of a cell scraper and lysed in sample buffer [60 mM Tris. HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1 mM dithiothreitol, 0.001% bromphenol blue] on ice for 20 minutes. Protein concentration was measured using protein assay reagents (Pierce). Twenty micrograms of protein were subjected to 14% SDS-PAGE and transferred to a nitrocellulose membrane. Each membrane was cut into two pieces. One piece, which contained proteins <30 kDa, was incubated with monoclonal p21 WAF1 antibody (1:1,000; Upstate) and the other with polyclonal betaactin antibody (1:2,000; Sigma Chemical), which served as a control for protein loading. The membranes were washed and incubated with secondary antibody conjugated with peroxidase, and the signal was detected using the chemiluminescent detection system (Pierce).

Protein Extraction and Western Blot Analysis for Bcl-2, Bax, and p53
H460 and H322 cells were treated with 30 and 50 muM genistein or sodium carbonate. Alter 24, 48, and 72 hours, the cells were harvested by scraping the cells from culture dishes and collected by centrifugation. Cells were resuspended in Tris. HCl buffer, sonicated twice for 10 seconds each, and lysed using an equal volume of 4% SDS. Protein concentration was then measured using protein assay reagents (Pierce). Twenty micrograms of protein were subjected to 14% or 10% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. Membranes were incubated with monoclonal Bcl-2, p53 (1:500; Oncogene), Bax (1:5,000; Biomol), and polyclonal beta-actin (1:2,000; Sigma Chemical) antibodies, washed with Tris-buffered saline-Tween 20, and incubated with secondary antibody conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Pierce).

Densitometric Analysis
Autoradiograms of the Western blots were scanned with a Gel Doc 1000 image scanner (Bio-Rad) that was linked to a Macintosh computer. The bidimensional optical densities of p21WAF1, Bcl, p53, Bcl-2, and actin proteins on the films were quantified and analyzed with Molecular Analyst software (Bio-Rad). The ratios of p21WAF1 to actin, Bax to actin, and p53 to actin were calculated with standardization of the ratios of each control to the unit value.

Results
Effects of Genistein on Cell Proliferation
To test the effects of genistein on growth of NSCLC in vitro, NSCLC cells were treated with 5, 10, 20, 30, and 50 muM genistein for 24-72 hours. This resulted in a dose-dependent inhibition of cell proliferation. The effects of genistein on the proliferation of H460 and H322 cells are depicted in Figure 1. Inhibition of cell proliferation could be the result of the induction of apoptosis, cell cycle growth arrest, and/or inhibition of growth. Therefore, we investigated whether genistein could induce apoptosis in NSCLC cells.

Induction of Apoptosis
After treatment with genistein, the possibility of an increase in the rate of cell death was suggested by the failure of cell numbers to increase with time. This is consistent with the hypothesis that genistein may induce apoptotic cell death. Previous reports have shown that treatment of human cancer cell lines with genistein did induce apoptosis ( 3, 11). Evidence of apoptosis was sought by first looking for the formation of nuclear apoptotic bodies. DAPI can bind specifically to nuclear DNA and emit a blue light visible with a fluorescence microscope. H460 and H322 cells in our experiment were grown in the presence of 50 muM genistein for various time periods and analyzed by DAPI staining for the formation of apoptotic bodies. We observed numerous apoptotic cells that showed nuclear condensation, formation of apoptotic bodies, giant cell formation, and fragmentation (Figure 2). Furthermore, 7-AAD staining and flow cytometry were also used to detect apoptosis in genistein-treated cells. The results showed that there was an increase in H460 apoptotic cells after genistein treatment for 48 hours (32.86%) and 72 hours (43.51%) compared with the control sample (17.98%) and that 94.08% and 96.84% of genistein-treated H322 cells entered apoptosis at two and three days, respectively, compared with 8% of the control group (Figure 3). The percentage of necrotic cells among the H322 control cells (Figure 3D) is higher, because the control ceils reached confluence after three days of incubation. Finally, genistein-induced apoptosis in H460 and H322 cells was also demonstrated by PARP assay (Figure 4). The full size of PARP protein (116 kDa) was degraded with the accumulation of the 85-kDa protein in cells after 48 hours of genistein treatment. These three independent methods of measuring apoptosis provided similar results, suggesting that genistein induces apoptosis in NSCLC cells in addition to cell growth inhibition. These results, however, do not provide any evidence regarding the mechanism by which genistein induces apoptosis. To explore such mechanisms, we investigated the alterations in the expression of the genes that are involved in the apoptotic pathway.

p53 Protein Expression During Genistein Treatment
The expression of p53 in H460 and H322 cells with and without genistein treatment was tested by Western blot analysis. It is important to note that H460 cells harbor wildtype p53 and H322 cells possess a mutation in the p53 gene (codon 248, CGG to CTG, Arg to Leu). As shown in Figure 5, an upregulation of p53 was detected in genistein-treated H460 cells compared with the control. To obtain a quantitative value for the protein expression of p53, optical density was measured (see Materials and Methods). The ratios of p53 to actin protein expression revealed that H460 cells treated with genistein showed at least a threefold increase in p53 compared with the untreated control (Figure 6). No change in p53 expression was observed in H322 cells that underwent the same genistein treatment (Figures 5 and 6).

Effect of Genistein on p21WAF1Expression
The expression of p21WAF1 in H460 and H322 cells was investigated in genistein-treated and untreated cells by Western blot analysis. The results of a typical experiment are shown in Figure 5, which demonstrates the induction of p21WAF1 protein when H460 and H322 cells were treated with 30 or 50 muM genistein after 24 hours. Optical density was also measured to obtain a quantitative value for the protein expression of p21WAF1. The ratios of p21WAF1 to actin protein expression revealed that H460 and H322 cells treated with genistein showed a two- to fivefold increase in p21WAF1 compared with the untreated control (Figure 6). The induction of p21WAF1 protein expression was directly correlated with the inhibition of cell growth.

Bax and Bcl-2 Protein Expression During Genistein Treatment
The constitutive levels of Bax or Bcl-2 and the time course for the effect of genistein on Bax or Bcl-2 expression in H460 and H322 cells were studied by Western blot analysis. The levels of Bax expression in H460 and H322 cells were upregulated with the addition of genistein after 24 hours (Figure 5). Optical density measurement showed a 4-fold increase in H460 cells and a 1.8-fold increase in H322 cells (Figure 6). In contrast, the expressions of Bclo2 remained unchanged after 24 hours of genistein treatment (Figures 5 and 6).

Discussion
Genistein, a PTK inhibitor and a topoisomerase II inhibitor, has multiple functions on tumor cell growth and differentiation. Experimental studies have shown the inhibition of cell growth of a wide range of cultured cancer cells, including leukemia, breast and prostate cancer, and lymphoma by genistein ( 2, 11, 12). Our data also demonstrated that genistein inhibits the growth of H460 and H322 NSCLC cell lines and that the inhibition is dose and time dependent. The decreased cell numbers in the treatment groups compared with the control group may be due to apoptotic cell death as well as inhibition of cell proliferation. DAPI labeling and cleavage analysis of PARP have been used as markers of apoptosis. Additionally, flow cytometric analysis with 7-AAD staining now has been conducted to detect and quantify apoptotic cells ( 22). Using these techniques, we, indeed, found induced apoptosis in the H460 and H322 cells treated with genistein. DAPI labeling showed that chromatin condensatiofi and nuclear fragmentation were present in H460 and H322 cells after 24-72 hours of genistein treatment. Accordingly, the apoptosis-associated proteolytic cleavage of PARP was detected in both cell lines treated with genistein. Flow-cytometric analysis revealed that the number of apoptotic cells increased with longer treatment period with genistein. These results suggest that genistein can inhibit the growth of NSCLC cells and induce apoptosis.

The product of the p53 gene, the tumor suppressor gene, is involved in many cellular processes, including gene transcription, DNA repair, genomic stability, cell cycle control, and apoptosis ( 23). The status of p53 is thought to be an important mediator in the cellular response to chemotherapy ( 24). Our results clearly demonstrate that H322 cells (containing mutant p53) and H460 cells (containing wildtype p53) are sensitive to genistein and proceed to apoptosis with genistein treatment. No correlation between p53 status and induction of apoptosis was detected, suggesting the presence of a p53-independent pathway through which genistein induces apoptosis in NSCLC. Previously, p53-independent apoptosis has been observed in a variety of tumor cell types ( 24). Although it is clear that chemotherapy- and radiation induced apoptosis can proceed, despite the absence of functional p53, little is known about the activation and signaling pathway involved in p53-independent apoptosis. Our data may imply that induction of apoptosis in NSCLC by genistein is p53 independent and is related to p21WAF1 induction. We previously reported that genistein can upregulate p21WAF1 and induceG2-M arrest and apoptosis in H460 cells ( 25). Shao and associates ( 26) also reported that genistein induced apoptosis and that cell cycle arrest is followed by increased p21WAF1 protein and mRNA and mediated through a p53-independent mechanism.

Members of the Bcl-2 protein family have been implicated as important components of the apoptosis pathway in a wide range of different cell types ( 27). Apoptosis is inhibited by Bcl-2 and Bcl-XL and promoted by Bcl-Xs and Bax ( 19, 20). Our data showed no change in Bcl-2 expression in NSCLC cells after treatment with genistein. The expression of Bax, however, was upregulated in NSCLC cells after genistein treatment for 24 hours. The ratios of Bax to actin protein expression revealed that H460 and H322 cells treated with genistein showed at least a 4- and a 1.8-fold increase in Bax compared with the untreated control. These results corresponded with a significant increase of apoptotic cells after 48 hours of genistein treatment. Bax harbors a high homologous sequence with Bcl-2 and forms heterodimers with Bcl-2. Thus Bax is a protein that antagonizes the antiapoptotic function of Bcl-2. Our results suggest that upregulation of Bax may, in part, be one of the molecular mechanisms through which genistein induces apoptosis. Bax is a transcriptional target of p53; however, our data showed no correlation between Bax and p53 in genistein-treated NSCLC cells. Further indepth studies are needed to establish the role of Bax in genistein-induced apoptosis.

In summary, our studies suggest that genistein inhibits cell growth of NSCLC and induces apoptosis through a p53-independent pathway with induction of Bax and p21WAF1 expression; however, further studies are needed to firmly establish the role of Bax and p21WAF1 in genistein-induced apoptosis in lung cancer. This characteristic of genistein, in conjunction with its nontoxic nature, could make it a potentially effective chemopreventive or therapeutic agent against NSCLC. Further in vivo studies, however, are needed to establish the role of genistein as a chemopreventive or therapeutic agent against NSCLC.

Acknowledgments and Notes
The authors thank Patricia Arlauskas for editorial assistance. Address reprint requests to Dr. Fazlul H. Sarkar, Dept. of Pathology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201.

Submitted 19 October 1998; accepted in final form 22 December 1998.

GRAPH: Figure 1. Effect of 0-50 muM genistein on growth of H460 (left) and H322 (right) non-small-cell lung cancer (NSCLC) cell lines.

GRAPH: Figure 2. Fluorescence microscopy of H460 [control (A) and treated (B)] and H322 [control (C) and treated (D)] cells incubated for 48 h and stained with 4,6-diamidino-2-phenylindole. Note normal nuclear pattern in control cells (A and C) and chromatin condensation and "apoptotic bodies" in genistein-treated cells (B and D).

GRAPH: Figure 3. Scattergrams of 7-amino actinomycin D-stained H460 (A-C) and H322 (D-E) cells. A and D: control; B and E: genistein treatment for 48 h; C and F: genistein treatment for 72 h.

GRAPH: Figure 4. Western blot analysis of poly(ADP-ribose) polymerase cleavage in H460 and H322 cells treated with genistein. -, Control; +, cells treated with genistein for 48 h.

GRAPH: Figure 5. Western blot analysis of p53, p21, Bc12, and Bax protein levels in H460 (left) and H322 cells (right) treated with 30 and 50 muM genistein. C, control.

GRAPH: Figure 6. Densitometric analysis of p53 (top), p21WAF1 (middle), and Bax (bottom) expression in H460 and H322 cells treated with genistein. OD, optical density.

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By Fangru Lian; Yiwei Li; Mahbubur Bhuiyan and Fazlul H. Sarkar

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