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Inhibition of cyclooxygenase-2 activity in head and neck cancer cells by genistein
Cancer Letters 211 (2004) 39–46 www.elsevier.com/locate/canlet
Inhibition of cyclooxygenase-2 activity in head and neck cancer cells by genistein Fei Ye, Josephine Wu, Trish Dunn, Jizu Yi, Xiaodi Tong, David Zhang* Department of Pathology, Mount Sinai School of Medicine, New York University, New York, NY 10029 USA Received 21 November 2003; received in revised form 19 March 2004; accepted 19 March 2004
Abstract Genistein, rich in soybean, has been reported to have anti-cancer activity on several cancers. However, the molecular mechanism of its anti-cancer activity still remains unclear. We investigated the effect of genistein on a human oral squamous carcinoma line (SCC-25), and demonstrated that genistein inhibited SCC-25 cell growth via G2/M phase arrest. We observed a significant decrease of proliferating cell nuclear antigen expression in these cells after treatment, but no significant change in the number of apoptotic cells, indicating that the major action of genistein is inhibition of cancer cell proliferation. We also observed a high level of prostaglandin E2 (PGE2) in these cells and PGE2 synthesis in SCC-25 cells was significantly suppressed by genistein. We demonstrated that genistein directly inhibited cycloxygenase-2 (COX-2) activity, an inducible enzyme that converts arachidonic acid to prostaglandins, similar to the action of celecoxib, a selective COX-2 inhibitor. However, the anticancer activity of genistein was much weaker than that of indomethacin (non-selective COX inhibitor), celecoxib and baicalein (flavonoid isolated from Scutellaria baicalensis). These results suggested that genistein might be useful as a chemopreventive agent rather than a chemotherapeutic agent. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Celecoxib; Indomethacin; Prostaglandin E2; Flavonoid
1. Introduction According to the report of the International Agency for Research on Cancer, approximately 170,000 men and 97,000 women worldwide have cancers of the oral cavity, and the most prevalent type of cancer is squamous cell carcinoma. In 2000, over 20,000 cases and nearly 5,000 deaths were reported in * Corresponding author. Address: Molecular Pathology Laboratory, Mount Sinai School of Medicine, P.O. Box 1122, One Gustave L. Levy Place, New York, NY 10021, USA. Tel.: þ 1212-659-8173; fax: þ 1-212-427-2082. E-mail address: [email protected] (D. Zhang).
North America, including the United States and Canada [1]. If oral cancer is treated in the early stage of tumor progression, the chance of cure is high. Unfortunately, most patients with this type of cancer have advanced disease at the time of diagnosis. Therefore, much research is currently devoted to chemotherapy and chemoprevention in order to treat or prevent oral cancers at an early stage. The arachidonic acid pathway is of particular importance to the etiology of head and neck squamous cell carcinoma (HNSCC). Arachidonic acid, a 20-carbon polyunsaturated fatty acid, is a phospholipid component of cell membranes. Two key
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.03.043
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enzymes in arachidonic acid metabolism are cyclooxygenase-1 (COX-1) and -2 (COX-2); the former is the constitutive isoform while the latter is inducible. An overproduction of COX-2 enzyme that catalyzes arachidonic acid metabolism to form prostaglandin E2 (PGE2) facilitates proliferation of neoplastic cells [2]. Patients with HNSCC have higher serum levels of PGE2 compared to healthy volunteers [3]. It has been shown that PGE2 was the predominant arachidonic acid metabolite in HNSCC [2]. Therefore, it is important to search for agents that inhibit the arachidonic acid pathway for the chemotherapeutic or chemopreventive treatment of HNSCC. Such work has lead to the discovery of current drugs including indomethacin that targets COX-1 and COX-2, and celecoxib that specifically targets the COX-2 enzyme. Genistein, one of the isoflavones rich in soybean and some forage plants, has been implied to decrease both the incidence and mortality in various cancers, including breast and prostate cancers [4,5]. In vitro studies demonstrated that genistein inhibits HNSCC cancer cell growth [6,7] and reduces PGE2 levels in mouse macrophages [8]. To further explore the mechanism of its anticancer activity, we investigated its effects on a human oral squamous carcinoma cell line, SCC-25. Our study revealed that genistein exhibited a significant inhibition on the growth of SCC-25 cells in parallel with the reduction of PCNA expression and PGE2 levels. These results suggested that genistein is a potential agent for cancer prevention.
bromide; Sigma, St. Louis, MO) assay to measure viable cells. Approximately 5 £ 103 cells were seeded onto each well of a 96-well plate for 24 h then treated with various concentrations of either genistein (Sigma) dissolved in 0.1 M Na2CO3, baicalein (Sigma) dissolved in 100% dimethyl sulfoxide (DMSO), indomethacin (Sigma) dissolved in 100% DMSO, or celecoxib (Searle, Skokie, IL) dissolved in 100% DMSO. The cells were then cultured for three days at 37 8C. After treatment, 10 ml of MTT at a concentration of 4 mg/ml was added to each well. The cells were incubated for an additional 4–6 h, and the supernatant was discarded. Finally, 100 ml of DMSO was added to the wells to dissolve the precipitate. Optical density (OD) was measured at a wavelength of 570 nm. 2.3. Cell cycle analysis 1.5 £ 105 cells/well were plated onto six-well plates and incubated for 24 h at 37 8C. Various concentrations of genistein were added to the wells and incubated for an additional 3 days. Cells were then washed, pelleted, fixed with cold 70% ethyl alcohol for at least 30 min, and incubated with 100 mg/ml RNase A and 50 mg/ml propidium iodide in phosphate buffer saline (PBS) at room temperature for 30 min. Samples were immediately analyzed by flow cytometry (Becton Dickinson, San Jose, CA). Cell cycle phase distribution was determined using Cell Quest Pro software (Becton Dickinson, Franklin Lakes, NJ). 2.4. Immunohistochemical staining
2. Experimental procedures 2.1. Cell culture SCC-25 cell line (American Type Culture Collection, Manassas, VA) was derived from tongue squamous cell carcinoma. The cells were incubated at 37 8C with a 5% CO2 atmosphere in a 50:50 mixture of DMEM and F12 containing 1% antibiotic/antimycotic, and 10% Fetal Bovine Serum. 2.2. Cell growth inhibition assay Growth inhibition of genistein was determined by an MTT (3, 4, 5-dimethylthiazol-2, 5-diphenyltetrazolium
Harvested cells were applied to polylysine-coated slides by centrifugation at 800 g for 5 min in a Cytospin centrifuge (Sakura Finetek USA, Inc., Torrance, CA). Cells were allowed to air dry, and fixed with 100% acetone. Slides were then incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity. After three washings with PBS, slides were incubated with blocking serum solution for 20 min. The solution was blotted off and 2 mg/ml of anti-proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added. Cells were then incubated overnight at 4 8C. After washing three times, the slides were incubated for 10 min with biotinylated
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second antibody (Zymed Lab. Inc., San Francisco, CA). Slides were washed three more times and then incubated for 10 min with streptavidin-peroxidase conjugate (Zymed Lab. Inc.). Color was developed by addition of diaminobenzoate chromogen (Zymed Lab. Inc.) and slides were counterstained with hematoxylin, followed by examination under a light microscope. Cells with a brown nuclear precipitation were considered PCNA positive whereas other cells staining blue were considered PCNA negative. The percentage of PCNA-positive cells was determined with a minimum of 500 cells per slide. 2.5. Apoptosis assay The detection of apoptotic cells was performed by simultaneous staining with both acridine orange (AO) (Sigma) and ethidium bromide (EB) (Sigma). Cells treated with various concentrations of agents for 72 h were collected by trypsinization and centrifugation. A quantity of 2 £ 105 pelleted cells were resuspended in 50 ml PBS containing 10 mg/ml of AO and 20 mg/ml of EB. Cells were then incubated in dark for 5 min at room temperature, mounted and observed under a fluorescence microscope with an FITC filter. EB stains dead cells and emits a red-orange fluorescence and AO stains live cells and emits apple green fluorescence. 2.6. PGE2 enzyme immunoassay A competitive PGE2 enzyme immunoassay was used to quantify the amount of PGE2 released from cancer cells into culture medium according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The supernatants of control and treated cell cultures were collected, centrifuged at 2500g for 2 min, and stored at 2 70 8C until tested. PGE2 standards were diluted with the appropriate media. The samples were added to appropriate wells and PGE2 conjugate was pipetted into all wells except the blank. Finally, monoclonal antibody against PGE2 was added to all wells except wells for blank and nonspecific binding, followed by incubation for 18 h at 4 8C. The plates were washed four times and color reaction was developed by the addition of tetramethylbenzidine substrate. After 30 min of incubation at room temperature,
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the reaction was quenched by addition of 1 M sulfuric acid. Optical density was measured at 450 nm on an ELX800 reader (Bio-Tek Instruments, Inc., Winooski, Vermont). In a parallel experiment, the total cells were counted under a light microscope. The PGE2 production was expressed as pg per 106 cells. The effect of genistein and celecoxib on COX-2 activity was further examined using an in-cell arachidonic acid conversion assay. The SCC-25 cells were incubated for 12 h and the medium was aspirated and replaced with fresh medium containing genistein (100 mM) or celecoxib (25 mM). The cells were further incubated for various periods of time (2, 4, 8, 12 and 24 h) to allow the agent to interact with COX-2 enzyme and the media were then aspirated. Fresh medium containing 100 mM arachidonic acid (Cayman Chemical Co. Ann Arbor, MI) was added and the cells were incubated for an additional 30 min to allow conversion of exogenous arachidonic acid to PGE2. An aliquot of medium (50 ul) was then removed and PGE2 was measured as described above. 2.7. Western blot analysis SCC-25 cells were treated with genistein and the proteins were extracted from the cells using a buffer containing 50 mM Tris – HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 1 £ protease inhibitors (Roche Applied Science; Indianapolis, IN). Fifteen micrograms of protein was fractionated by electrophoresis through a 10% SDS polyacrylamide gel and the proteins then transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h in a blocking buffer containing 5% dry milk, 20 mM Tris – HCl (pH 7.5), 100 mM NaCl and 0.1% Tween-20 and then incubated with a polyclonal anti-COX-2 antibody (1:400 dilution, Cayman Chemical Co.) in the blocking buffer at 4 8C overnight. The membrane was then incubated with an anti-rabbit antibody conjugated with horseradish peroxidase (Amersham, Arlington Height, IL) and the protein was detected using a chemiluminescence method followed by autoradiography. The same membrane was then used to detect b-actin protein using a monoclonal anti-b-actin antibody (1:10,000 dilution, Sigma) as described above.
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3. Results 3.1. Genistein inhibits cell growth We investigated the anti-cancer activity of genistein and compared it with indomethacin (a nonselective COX inhibitor), celecoxib (a COX-2 specific inhibitor), and baicalein (an isoflavone isolated from an herbal medicine, Scutellaria baicalensis). Our results show that all of four agents were capable of inhibiting the growth of human oral squamous carcinoma cells (SCC-25) (Fig. 1). Among these four agents, celecoxib exerted the strongest inhibitory effect on SCC-25 cells with a 50% inhibition concentration (IC50) of 20 mM. Indomethacin and baicalein exerted a similar inhibitory effect on these cells with an IC50 of 70 mM. Genistein also exerted an inhibitory effect on SCC-25 cells although its effect was weaker than those of other three agents. The IC50 for genistein was , 200 mM, which was 10-fold higher than that of celecoxib (Fig. 1). We further analyzed the effect of genistein on cell cycles. Our results show that genistein caused a significant G2/M phase arrest with concurrent decrease of G0/G1 phase (Fig. 2).
and other agents. Our results show that no significant increase of apoptotic cells was seen in the presence of 50 mM genistein (3% apoptotic cells in control and 5% in treated cells). No significant increase of apoptotic cells was seen in celecoxib and indomethacin treated cells at 10 mM. However, baicalein induced a slight increase of apoptosis (8%) in SCC-25 cells at 10 mM. 3.4. Genistein inhibited the PGE2 level in cancer cells Since there is a significant increase of PGE2 in HNSCC, we also measured the amount of PGE2 in SCC-25 cells and tested the effect of genistein on PGE2 production. Our results show that SCC-25 cells produced a high level of PGE2 (185 pg/106 cells) (Fig. 4A), supporting previously reported studies [9 –11]. The amount of PGE2 produced by these cells was decreased with the increase of genistein
3.2. Genistein reduces PCNA expression We measured the expression of PCNA, an indicator of cell proliferation potential, in SCC-25 cells. Our results show that SCC-25 cells express a high level of PCNA (60 –70% PCNA positive cells), indicating a high level of proliferative activity (Fig. 3). There was a dose-dependent decrease in the number of PCNA-positive SCC-25 cells. At 10 mM of genistein, the percentage of PCNA-positive cells decreased from 62 to 40%, which was statistically significant ðP , 0:01Þ: However, compared with the other three agents, genistein was weaker than celecoxib and indomethacin but stronger than baicalein. It is worthy to note that suppression of PCNA expression occurred before evidence of growth inhibition since no growth inhibition was observed at 10 mM of any agent (Fig. 1). 3.3. Effect of genistein on apoptosis To investigate if genistein can cause programmed cell death, we tested apoptosis induced by genistein
Fig. 1. Inhibition of HNSCC cell growth by genistein, baicalein, celecoxib, and indomethacin. 5 £ 103 cells/well were seeded onto a 96-well plate and were treated with various agents at different concentrations and growth inhibition was determined by MTT assay after 72-h treatment. Dose-dependent inhibition of cell growth was observed in SCC-25 cells after treatment with genistein, baicalein, indomethacin, and celecoxib at concentrations ranging from 5 – 400 mM. Each point represents the mean value ^ SD in quadruplicate.
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Fig. 2. Cell cycle analysis by flow cytometry: The SCC-25 cells were treated with 0, 50, 100, and 200 mM of genistein for 72 h, harvested and subjected to flow cytometry analysis. The histograms show a significant increase of a G2/M population with concomitant decrease of G0/G1 population after treatment with genistein.
concentration (Fig. 4A). A significant decrease in PGE2 was observed at 0.1 mM of genistein, and at which concentration, no significant growth inhibition was noted (Fig. 1). These results indicated that PGE2 is important in supporting cancer cell growth and decrease of PGE2 precedes the growth inhibition. We also compared the ability of other agents to inhibit PGE 2 synthesis and found that celecoxib and indomethacin significantly reduced PGE2 level even at 0.1 mM, while baicalein does not affect PGE2 synthesis at all (data not shown). These results indicated the increase of PGE2 production is due to an increase of COX-2 activity and/or expression. Furthermore, not all plant flavonoids can act on COX2: genistein reduces PGE2 synthesis but baicalein does not. However, the level of reduction of PGE2 for celecoxib and indomethacin was much greater: a 68% reduction in treated cells at 0.1 mM of both agents [11] vs. a 17% reduction at 0.1 mM of genistein (Fig. 4A). These results indicate that genistein is a weaker inhibitor of COX and this could explain its weaker anti-cancer activity.
Fig. 3. Suppression of PCNA expression by genistein, baicalein, celecoxib, and indomethacin. SCC-25 cells were treated with various agents for 72 h and the percentage of cells staining positive for PCNA were determined (500 cells were counted). Significant decreases of PNCA-positive cells were observed after treatment with indomethacin and celecoxib at concentrations of 0.1–10 mM. However, genistein and baicalein was less effective in suppressing PCNA expression. Results are mean values ^ SD (bars) of independent experiments performed in triplicate. The student’s t test was used to analyze the data. Asterisks indicate statistical significance ðP , 0:01Þ:
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Fig. 4. Inhibition of PGE2 synthesis by genistein. The amount of PGE2 present in the supernatant of the culture medium was measured by an enzyme immunoassay and expressed as pg per 106 cells or pg/ml. (A) In the presence of genistein at concentrations ranging from 0.1–100 mM, SCC-25 cells demonstrated a dose-dependant suppression of PGE2 synthesis after 12-h incubation. Results are mean values ^ SD (bars) of independent experiments performed in triplicate. The student’s t test was used to analyze the data. Asterisks indicate statistical significance ðP , 0:01Þ: (B) Suppression of in-cell conversion of arachidonic acid to PGE2 by genistein (100 mM) and celecoxib (25 mM). A rapid inhibition (2 h) was observed for both genistein and celecoxib, indicating direct inhibition of COX-2 activity by both agents.
We also examined the effect of genistein and celecoxib on COX-2 activity using an in-cell arachidonic acid conversion assay. By supplementing culture media with exogenous arachidonic acid, the substrate of COX-2, the effect of arachidonic acid release from membrane (i.e. protein kinase and phospholipase A2 activities) is eliminated [12]. Our results show that there is a significant decrease of PGE2 level in the presence of both genistein (100 mM) and celecoxib (25 mM) and kinetics for both agents were the same (Fig. 4B), indicating a similar mode of action of both agents.
3.5. Inhibition of COX-2 protein expression We further investigated the effect of genistein on COX-2 protein expression in SCC-25 cells using Western blotting. Our results showed that genistein does not affect COX-2 expression up to 72 h at concentrations as high as 100 mM (Fig. 5). These results supported the observation that genistein directly inhibits COX-2 activity.
Fig. 5. Analysis of COX-2 protein expression by Western blot. (A) SCC-25 cells were treated with genistein at 50 mM for 12, 24, 48, and 72 h. Proteins were extracted from cells and separated through SDS-PAGE. No decrease of COX-2 protein expression was observed up to 72-h treatment with genistein (upper panel). b-actin expression was used as an internal control (lower panel). (B) SCC-25 cells were treated with genistein at concentrations of 0, 10, 25, 50, and 100 mM for 72 h and the amount of COX-2 proteins were determined as above. No decrease of COX-2 protein expression was observed after treatment with genistein (upper panel). b-actin expression was used as an internal control (lower panel).
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4. Discussion Extensive work on the cancer-treating potential of genistein has been reported since a workshop at the National Cancer Institute on the observed anticarcinogenic properties of soybeans in 1990 [13]. Subsequent epidemiological studies conducted on the effects of soy intake on populations revealed that in China and Japan, where soy consumption is high, incidences of breast, colon, and prostate cancers were lower than those in Western nations [14,15]. There are also much lower occurrences of oral cancer mortality in the Japanese compared to Caucasian men and women in the US [16]. However, HNSCC remains a problem in Asian countries because of risk factors such as smoking, drinking, and chewing of betel quid or areca nut in these countries [17 –20]. Moderation in alcohol consumption and cessation of smoking in combination with dietary factors, especially genistein because of its antioxidant properties [18,21], may have an important role in HNSCC prevention [18,22]. A few reports showed that genistein has in vitro anticancer activity on various cancer cell lines including prostate and breast cancers [7,23]. It has been shown that genistein has several biological and biochemical effects, which may explain its anticancer activity. These activities include induction of apoptosis, inhibition of angiogenesis, inhibition of DNA topoisomerase II and inhibition of protein tyrosine kinases [24,25]. In addition, it has been shown that genistein down-regulates nuclear factor-kB and transforming growth factor-b and inhibits epidermal growth factor-induced growth [26]. Therefore, multiple mechanisms may operate together in a synergistic fashion. In this study, we demonstrated the growth of head and neck cancer cell line, SCC-25, was suppressed after treatment with genistein. These results are consistent with previous reports that genistein inhibits HNSCC cell proliferation [6,27]. However, genistein inhibits cell growth to a much lesser extent compared to other COX inhibitors (indomethacin and celecoxib) and a flavonoid isolated from other plants (baicalein) (Fig. 1). Furthermore, the inhibition concentration was above the physiological concentration (4 mM) of genistein in blood [28] and the active aglycone form of genistein could be even lower [29]. Therefore, it is unlikely that genistein can reach a blood concentration
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that can inhibit cancer cell growth at concentrations demonstrated in this study. However, recent animal studies demonstrated the concentration of the aglycone form of genistein in tissue is higher than in blood [29]. Therefore, the aglycone form of genistein in tumor tissues may reach a biologically effective level to inhibit cancer cell growth. On the other hand, the effect of genistein on PCNA and PGE2 were observed at concentrations within the physiological range, also suggesting its role in cancer prevention. The major action of genistein on HNSCC is to inhibit cell proliferation at the G2/M phase (Fig. 2), similar to the action of baicalein (data not shown). In contrast, celecoxib and indomethacin caused a significant arrest at the G0/G1 phase [11]. These results indicated that different mechanisms of cell growth inhibition exist between genistein and celecoxib although both inhibit PGE2 synthesis. One of the mechanisms of cell cycle arrest caused by genistein is up-regulation of the cell cycle regulatory molecule p21WAF, which can form a complex with PCNA that is also inhibited by genistein (Fig. 3). Changes in these two important cell cycle regulatory proteins result in decrease of kinase activity of the Cyclin B1-Cdk1 complex and G2/M arrest [6]. In our study, we found little evidence of induction of apoptosis in SCC-25 cells after treatment with genistein. In addition, we did not observe any morphological changes indicative of differentiation in genistein-treated cells. This is contradictory to a previous report by Alhasan et al. [6], that showed cancer cells to be more rounded and detached after treatment with genistein and more apoptotic bodies were observed. These differences may be due to different cell lines used in these studies, indicating heterogeneity of HNSCC. We demonstrated that genistein causes a decrease in the production of PGE2 in SCC-25 cells even at a low concentration, which parallels a decrease of PCNA expression. Increased PGE2 levels found in HNSCC cell lines, suggested its role in enhancing cancer cell growth [9,10]. It has been shown that PGE2 may regulate cancer cell proliferation in an autocrine and/or paracrine manner via the prostaglandin receptors, especially EP2 [30]. Stimulation of the receptor resulted in an increase of cAMP levels in cancer cells that triggers the signal transduction pathway [31]. PGE2 can also bind to prostaglandin receptor EP1,
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a nuclear protein, which can increase gene expression leading to uncontrolled cell proliferation. We further investigated the effect of genistein on COX-2 expression and activity. Our results showed that genistein exerted no effect on COX-2 protein expression (Fig. 5). However, it directly inhibited COX-2 enzymatic activity, similar to the action of celecoxib, a COX-2 specific inhibitor (Fig. 4B). Compared to celecoxib it was less effective, indicating that genistein may bind to COX-2 at a different site from celecoxib or with weaker affinity [32]. In summary, genistein exerts some anticancer activity through the action of decreasing cell proliferation. This activity may be related to suppression of PCNA expression and reduction of PGE2 synthesis via inhibition of COX-2 activity. However, its anticancer activity is much weaker compared to other COX specific inhibitors and flavonoids isolated from other plants. Therefore, genistein may be more useful in chemoprevention rather than chemotherapy for HNSCC.
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Inhibition of cyclooxygenase-2 activity in head and neck cancer cells by genistein Fei Ye, Josephine Wu, Trish Dunn, Jizu Yi, Xiaodi Tong, David Zhang* Department of Pathology, Mount Sinai School of Medicine, New York University, New York, NY 10029 USA Received 21 November 2003; received in revised form 19 March 2004; accepted 19 March 2004
Abstract Genistein, rich in soybean, has been reported to have anti-cancer activity on several cancers. However, the molecular mechanism of its anti-cancer activity still remains unclear. We investigated the effect of genistein on a human oral squamous carcinoma line (SCC-25), and demonstrated that genistein inhibited SCC-25 cell growth via G2/M phase arrest. We observed a significant decrease of proliferating cell nuclear antigen expression in these cells after treatment, but no significant change in the number of apoptotic cells, indicating that the major action of genistein is inhibition of cancer cell proliferation. We also observed a high level of prostaglandin E2 (PGE2) in these cells and PGE2 synthesis in SCC-25 cells was significantly suppressed by genistein. We demonstrated that genistein directly inhibited cycloxygenase-2 (COX-2) activity, an inducible enzyme that converts arachidonic acid to prostaglandins, similar to the action of celecoxib, a selective COX-2 inhibitor. However, the anticancer activity of genistein was much weaker than that of indomethacin (non-selective COX inhibitor), celecoxib and baicalein (flavonoid isolated from Scutellaria baicalensis). These results suggested that genistein might be useful as a chemopreventive agent rather than a chemotherapeutic agent. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Celecoxib; Indomethacin; Prostaglandin E2; Flavonoid
1. Introduction According to the report of the International Agency for Research on Cancer, approximately 170,000 men and 97,000 women worldwide have cancers of the oral cavity, and the most prevalent type of cancer is squamous cell carcinoma. In 2000, over 20,000 cases and nearly 5,000 deaths were reported in * Corresponding author. Address: Molecular Pathology Laboratory, Mount Sinai School of Medicine, P.O. Box 1122, One Gustave L. Levy Place, New York, NY 10021, USA. Tel.: þ 1212-659-8173; fax: þ 1-212-427-2082. E-mail address: [email protected] (D. Zhang).
North America, including the United States and Canada [1]. If oral cancer is treated in the early stage of tumor progression, the chance of cure is high. Unfortunately, most patients with this type of cancer have advanced disease at the time of diagnosis. Therefore, much research is currently devoted to chemotherapy and chemoprevention in order to treat or prevent oral cancers at an early stage. The arachidonic acid pathway is of particular importance to the etiology of head and neck squamous cell carcinoma (HNSCC). Arachidonic acid, a 20-carbon polyunsaturated fatty acid, is a phospholipid component of cell membranes. Two key
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.03.043
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enzymes in arachidonic acid metabolism are cyclooxygenase-1 (COX-1) and -2 (COX-2); the former is the constitutive isoform while the latter is inducible. An overproduction of COX-2 enzyme that catalyzes arachidonic acid metabolism to form prostaglandin E2 (PGE2) facilitates proliferation of neoplastic cells [2]. Patients with HNSCC have higher serum levels of PGE2 compared to healthy volunteers [3]. It has been shown that PGE2 was the predominant arachidonic acid metabolite in HNSCC [2]. Therefore, it is important to search for agents that inhibit the arachidonic acid pathway for the chemotherapeutic or chemopreventive treatment of HNSCC. Such work has lead to the discovery of current drugs including indomethacin that targets COX-1 and COX-2, and celecoxib that specifically targets the COX-2 enzyme. Genistein, one of the isoflavones rich in soybean and some forage plants, has been implied to decrease both the incidence and mortality in various cancers, including breast and prostate cancers [4,5]. In vitro studies demonstrated that genistein inhibits HNSCC cancer cell growth [6,7] and reduces PGE2 levels in mouse macrophages [8]. To further explore the mechanism of its anticancer activity, we investigated its effects on a human oral squamous carcinoma cell line, SCC-25. Our study revealed that genistein exhibited a significant inhibition on the growth of SCC-25 cells in parallel with the reduction of PCNA expression and PGE2 levels. These results suggested that genistein is a potential agent for cancer prevention.
bromide; Sigma, St. Louis, MO) assay to measure viable cells. Approximately 5 £ 103 cells were seeded onto each well of a 96-well plate for 24 h then treated with various concentrations of either genistein (Sigma) dissolved in 0.1 M Na2CO3, baicalein (Sigma) dissolved in 100% dimethyl sulfoxide (DMSO), indomethacin (Sigma) dissolved in 100% DMSO, or celecoxib (Searle, Skokie, IL) dissolved in 100% DMSO. The cells were then cultured for three days at 37 8C. After treatment, 10 ml of MTT at a concentration of 4 mg/ml was added to each well. The cells were incubated for an additional 4–6 h, and the supernatant was discarded. Finally, 100 ml of DMSO was added to the wells to dissolve the precipitate. Optical density (OD) was measured at a wavelength of 570 nm. 2.3. Cell cycle analysis 1.5 £ 105 cells/well were plated onto six-well plates and incubated for 24 h at 37 8C. Various concentrations of genistein were added to the wells and incubated for an additional 3 days. Cells were then washed, pelleted, fixed with cold 70% ethyl alcohol for at least 30 min, and incubated with 100 mg/ml RNase A and 50 mg/ml propidium iodide in phosphate buffer saline (PBS) at room temperature for 30 min. Samples were immediately analyzed by flow cytometry (Becton Dickinson, San Jose, CA). Cell cycle phase distribution was determined using Cell Quest Pro software (Becton Dickinson, Franklin Lakes, NJ). 2.4. Immunohistochemical staining
2. Experimental procedures 2.1. Cell culture SCC-25 cell line (American Type Culture Collection, Manassas, VA) was derived from tongue squamous cell carcinoma. The cells were incubated at 37 8C with a 5% CO2 atmosphere in a 50:50 mixture of DMEM and F12 containing 1% antibiotic/antimycotic, and 10% Fetal Bovine Serum. 2.2. Cell growth inhibition assay Growth inhibition of genistein was determined by an MTT (3, 4, 5-dimethylthiazol-2, 5-diphenyltetrazolium
Harvested cells were applied to polylysine-coated slides by centrifugation at 800 g for 5 min in a Cytospin centrifuge (Sakura Finetek USA, Inc., Torrance, CA). Cells were allowed to air dry, and fixed with 100% acetone. Slides were then incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity. After three washings with PBS, slides were incubated with blocking serum solution for 20 min. The solution was blotted off and 2 mg/ml of anti-proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added. Cells were then incubated overnight at 4 8C. After washing three times, the slides were incubated for 10 min with biotinylated
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second antibody (Zymed Lab. Inc., San Francisco, CA). Slides were washed three more times and then incubated for 10 min with streptavidin-peroxidase conjugate (Zymed Lab. Inc.). Color was developed by addition of diaminobenzoate chromogen (Zymed Lab. Inc.) and slides were counterstained with hematoxylin, followed by examination under a light microscope. Cells with a brown nuclear precipitation were considered PCNA positive whereas other cells staining blue were considered PCNA negative. The percentage of PCNA-positive cells was determined with a minimum of 500 cells per slide. 2.5. Apoptosis assay The detection of apoptotic cells was performed by simultaneous staining with both acridine orange (AO) (Sigma) and ethidium bromide (EB) (Sigma). Cells treated with various concentrations of agents for 72 h were collected by trypsinization and centrifugation. A quantity of 2 £ 105 pelleted cells were resuspended in 50 ml PBS containing 10 mg/ml of AO and 20 mg/ml of EB. Cells were then incubated in dark for 5 min at room temperature, mounted and observed under a fluorescence microscope with an FITC filter. EB stains dead cells and emits a red-orange fluorescence and AO stains live cells and emits apple green fluorescence. 2.6. PGE2 enzyme immunoassay A competitive PGE2 enzyme immunoassay was used to quantify the amount of PGE2 released from cancer cells into culture medium according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The supernatants of control and treated cell cultures were collected, centrifuged at 2500g for 2 min, and stored at 2 70 8C until tested. PGE2 standards were diluted with the appropriate media. The samples were added to appropriate wells and PGE2 conjugate was pipetted into all wells except the blank. Finally, monoclonal antibody against PGE2 was added to all wells except wells for blank and nonspecific binding, followed by incubation for 18 h at 4 8C. The plates were washed four times and color reaction was developed by the addition of tetramethylbenzidine substrate. After 30 min of incubation at room temperature,
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the reaction was quenched by addition of 1 M sulfuric acid. Optical density was measured at 450 nm on an ELX800 reader (Bio-Tek Instruments, Inc., Winooski, Vermont). In a parallel experiment, the total cells were counted under a light microscope. The PGE2 production was expressed as pg per 106 cells. The effect of genistein and celecoxib on COX-2 activity was further examined using an in-cell arachidonic acid conversion assay. The SCC-25 cells were incubated for 12 h and the medium was aspirated and replaced with fresh medium containing genistein (100 mM) or celecoxib (25 mM). The cells were further incubated for various periods of time (2, 4, 8, 12 and 24 h) to allow the agent to interact with COX-2 enzyme and the media were then aspirated. Fresh medium containing 100 mM arachidonic acid (Cayman Chemical Co. Ann Arbor, MI) was added and the cells were incubated for an additional 30 min to allow conversion of exogenous arachidonic acid to PGE2. An aliquot of medium (50 ul) was then removed and PGE2 was measured as described above. 2.7. Western blot analysis SCC-25 cells were treated with genistein and the proteins were extracted from the cells using a buffer containing 50 mM Tris – HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 1 £ protease inhibitors (Roche Applied Science; Indianapolis, IN). Fifteen micrograms of protein was fractionated by electrophoresis through a 10% SDS polyacrylamide gel and the proteins then transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h in a blocking buffer containing 5% dry milk, 20 mM Tris – HCl (pH 7.5), 100 mM NaCl and 0.1% Tween-20 and then incubated with a polyclonal anti-COX-2 antibody (1:400 dilution, Cayman Chemical Co.) in the blocking buffer at 4 8C overnight. The membrane was then incubated with an anti-rabbit antibody conjugated with horseradish peroxidase (Amersham, Arlington Height, IL) and the protein was detected using a chemiluminescence method followed by autoradiography. The same membrane was then used to detect b-actin protein using a monoclonal anti-b-actin antibody (1:10,000 dilution, Sigma) as described above.
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3. Results 3.1. Genistein inhibits cell growth We investigated the anti-cancer activity of genistein and compared it with indomethacin (a nonselective COX inhibitor), celecoxib (a COX-2 specific inhibitor), and baicalein (an isoflavone isolated from an herbal medicine, Scutellaria baicalensis). Our results show that all of four agents were capable of inhibiting the growth of human oral squamous carcinoma cells (SCC-25) (Fig. 1). Among these four agents, celecoxib exerted the strongest inhibitory effect on SCC-25 cells with a 50% inhibition concentration (IC50) of 20 mM. Indomethacin and baicalein exerted a similar inhibitory effect on these cells with an IC50 of 70 mM. Genistein also exerted an inhibitory effect on SCC-25 cells although its effect was weaker than those of other three agents. The IC50 for genistein was , 200 mM, which was 10-fold higher than that of celecoxib (Fig. 1). We further analyzed the effect of genistein on cell cycles. Our results show that genistein caused a significant G2/M phase arrest with concurrent decrease of G0/G1 phase (Fig. 2).
and other agents. Our results show that no significant increase of apoptotic cells was seen in the presence of 50 mM genistein (3% apoptotic cells in control and 5% in treated cells). No significant increase of apoptotic cells was seen in celecoxib and indomethacin treated cells at 10 mM. However, baicalein induced a slight increase of apoptosis (8%) in SCC-25 cells at 10 mM. 3.4. Genistein inhibited the PGE2 level in cancer cells Since there is a significant increase of PGE2 in HNSCC, we also measured the amount of PGE2 in SCC-25 cells and tested the effect of genistein on PGE2 production. Our results show that SCC-25 cells produced a high level of PGE2 (185 pg/106 cells) (Fig. 4A), supporting previously reported studies [9 –11]. The amount of PGE2 produced by these cells was decreased with the increase of genistein
3.2. Genistein reduces PCNA expression We measured the expression of PCNA, an indicator of cell proliferation potential, in SCC-25 cells. Our results show that SCC-25 cells express a high level of PCNA (60 –70% PCNA positive cells), indicating a high level of proliferative activity (Fig. 3). There was a dose-dependent decrease in the number of PCNA-positive SCC-25 cells. At 10 mM of genistein, the percentage of PCNA-positive cells decreased from 62 to 40%, which was statistically significant ðP , 0:01Þ: However, compared with the other three agents, genistein was weaker than celecoxib and indomethacin but stronger than baicalein. It is worthy to note that suppression of PCNA expression occurred before evidence of growth inhibition since no growth inhibition was observed at 10 mM of any agent (Fig. 1). 3.3. Effect of genistein on apoptosis To investigate if genistein can cause programmed cell death, we tested apoptosis induced by genistein
Fig. 1. Inhibition of HNSCC cell growth by genistein, baicalein, celecoxib, and indomethacin. 5 £ 103 cells/well were seeded onto a 96-well plate and were treated with various agents at different concentrations and growth inhibition was determined by MTT assay after 72-h treatment. Dose-dependent inhibition of cell growth was observed in SCC-25 cells after treatment with genistein, baicalein, indomethacin, and celecoxib at concentrations ranging from 5 – 400 mM. Each point represents the mean value ^ SD in quadruplicate.
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Fig. 2. Cell cycle analysis by flow cytometry: The SCC-25 cells were treated with 0, 50, 100, and 200 mM of genistein for 72 h, harvested and subjected to flow cytometry analysis. The histograms show a significant increase of a G2/M population with concomitant decrease of G0/G1 population after treatment with genistein.
concentration (Fig. 4A). A significant decrease in PGE2 was observed at 0.1 mM of genistein, and at which concentration, no significant growth inhibition was noted (Fig. 1). These results indicated that PGE2 is important in supporting cancer cell growth and decrease of PGE2 precedes the growth inhibition. We also compared the ability of other agents to inhibit PGE 2 synthesis and found that celecoxib and indomethacin significantly reduced PGE2 level even at 0.1 mM, while baicalein does not affect PGE2 synthesis at all (data not shown). These results indicated the increase of PGE2 production is due to an increase of COX-2 activity and/or expression. Furthermore, not all plant flavonoids can act on COX2: genistein reduces PGE2 synthesis but baicalein does not. However, the level of reduction of PGE2 for celecoxib and indomethacin was much greater: a 68% reduction in treated cells at 0.1 mM of both agents [11] vs. a 17% reduction at 0.1 mM of genistein (Fig. 4A). These results indicate that genistein is a weaker inhibitor of COX and this could explain its weaker anti-cancer activity.
Fig. 3. Suppression of PCNA expression by genistein, baicalein, celecoxib, and indomethacin. SCC-25 cells were treated with various agents for 72 h and the percentage of cells staining positive for PCNA were determined (500 cells were counted). Significant decreases of PNCA-positive cells were observed after treatment with indomethacin and celecoxib at concentrations of 0.1–10 mM. However, genistein and baicalein was less effective in suppressing PCNA expression. Results are mean values ^ SD (bars) of independent experiments performed in triplicate. The student’s t test was used to analyze the data. Asterisks indicate statistical significance ðP , 0:01Þ:
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Fig. 4. Inhibition of PGE2 synthesis by genistein. The amount of PGE2 present in the supernatant of the culture medium was measured by an enzyme immunoassay and expressed as pg per 106 cells or pg/ml. (A) In the presence of genistein at concentrations ranging from 0.1–100 mM, SCC-25 cells demonstrated a dose-dependant suppression of PGE2 synthesis after 12-h incubation. Results are mean values ^ SD (bars) of independent experiments performed in triplicate. The student’s t test was used to analyze the data. Asterisks indicate statistical significance ðP , 0:01Þ: (B) Suppression of in-cell conversion of arachidonic acid to PGE2 by genistein (100 mM) and celecoxib (25 mM). A rapid inhibition (2 h) was observed for both genistein and celecoxib, indicating direct inhibition of COX-2 activity by both agents.
We also examined the effect of genistein and celecoxib on COX-2 activity using an in-cell arachidonic acid conversion assay. By supplementing culture media with exogenous arachidonic acid, the substrate of COX-2, the effect of arachidonic acid release from membrane (i.e. protein kinase and phospholipase A2 activities) is eliminated [12]. Our results show that there is a significant decrease of PGE2 level in the presence of both genistein (100 mM) and celecoxib (25 mM) and kinetics for both agents were the same (Fig. 4B), indicating a similar mode of action of both agents.
3.5. Inhibition of COX-2 protein expression We further investigated the effect of genistein on COX-2 protein expression in SCC-25 cells using Western blotting. Our results showed that genistein does not affect COX-2 expression up to 72 h at concentrations as high as 100 mM (Fig. 5). These results supported the observation that genistein directly inhibits COX-2 activity.
Fig. 5. Analysis of COX-2 protein expression by Western blot. (A) SCC-25 cells were treated with genistein at 50 mM for 12, 24, 48, and 72 h. Proteins were extracted from cells and separated through SDS-PAGE. No decrease of COX-2 protein expression was observed up to 72-h treatment with genistein (upper panel). b-actin expression was used as an internal control (lower panel). (B) SCC-25 cells were treated with genistein at concentrations of 0, 10, 25, 50, and 100 mM for 72 h and the amount of COX-2 proteins were determined as above. No decrease of COX-2 protein expression was observed after treatment with genistein (upper panel). b-actin expression was used as an internal control (lower panel).
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4. Discussion Extensive work on the cancer-treating potential of genistein has been reported since a workshop at the National Cancer Institute on the observed anticarcinogenic properties of soybeans in 1990 [13]. Subsequent epidemiological studies conducted on the effects of soy intake on populations revealed that in China and Japan, where soy consumption is high, incidences of breast, colon, and prostate cancers were lower than those in Western nations [14,15]. There are also much lower occurrences of oral cancer mortality in the Japanese compared to Caucasian men and women in the US [16]. However, HNSCC remains a problem in Asian countries because of risk factors such as smoking, drinking, and chewing of betel quid or areca nut in these countries [17 –20]. Moderation in alcohol consumption and cessation of smoking in combination with dietary factors, especially genistein because of its antioxidant properties [18,21], may have an important role in HNSCC prevention [18,22]. A few reports showed that genistein has in vitro anticancer activity on various cancer cell lines including prostate and breast cancers [7,23]. It has been shown that genistein has several biological and biochemical effects, which may explain its anticancer activity. These activities include induction of apoptosis, inhibition of angiogenesis, inhibition of DNA topoisomerase II and inhibition of protein tyrosine kinases [24,25]. In addition, it has been shown that genistein down-regulates nuclear factor-kB and transforming growth factor-b and inhibits epidermal growth factor-induced growth [26]. Therefore, multiple mechanisms may operate together in a synergistic fashion. In this study, we demonstrated the growth of head and neck cancer cell line, SCC-25, was suppressed after treatment with genistein. These results are consistent with previous reports that genistein inhibits HNSCC cell proliferation [6,27]. However, genistein inhibits cell growth to a much lesser extent compared to other COX inhibitors (indomethacin and celecoxib) and a flavonoid isolated from other plants (baicalein) (Fig. 1). Furthermore, the inhibition concentration was above the physiological concentration (4 mM) of genistein in blood [28] and the active aglycone form of genistein could be even lower [29]. Therefore, it is unlikely that genistein can reach a blood concentration
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that can inhibit cancer cell growth at concentrations demonstrated in this study. However, recent animal studies demonstrated the concentration of the aglycone form of genistein in tissue is higher than in blood [29]. Therefore, the aglycone form of genistein in tumor tissues may reach a biologically effective level to inhibit cancer cell growth. On the other hand, the effect of genistein on PCNA and PGE2 were observed at concentrations within the physiological range, also suggesting its role in cancer prevention. The major action of genistein on HNSCC is to inhibit cell proliferation at the G2/M phase (Fig. 2), similar to the action of baicalein (data not shown). In contrast, celecoxib and indomethacin caused a significant arrest at the G0/G1 phase [11]. These results indicated that different mechanisms of cell growth inhibition exist between genistein and celecoxib although both inhibit PGE2 synthesis. One of the mechanisms of cell cycle arrest caused by genistein is up-regulation of the cell cycle regulatory molecule p21WAF, which can form a complex with PCNA that is also inhibited by genistein (Fig. 3). Changes in these two important cell cycle regulatory proteins result in decrease of kinase activity of the Cyclin B1-Cdk1 complex and G2/M arrest [6]. In our study, we found little evidence of induction of apoptosis in SCC-25 cells after treatment with genistein. In addition, we did not observe any morphological changes indicative of differentiation in genistein-treated cells. This is contradictory to a previous report by Alhasan et al. [6], that showed cancer cells to be more rounded and detached after treatment with genistein and more apoptotic bodies were observed. These differences may be due to different cell lines used in these studies, indicating heterogeneity of HNSCC. We demonstrated that genistein causes a decrease in the production of PGE2 in SCC-25 cells even at a low concentration, which parallels a decrease of PCNA expression. Increased PGE2 levels found in HNSCC cell lines, suggested its role in enhancing cancer cell growth [9,10]. It has been shown that PGE2 may regulate cancer cell proliferation in an autocrine and/or paracrine manner via the prostaglandin receptors, especially EP2 [30]. Stimulation of the receptor resulted in an increase of cAMP levels in cancer cells that triggers the signal transduction pathway [31]. PGE2 can also bind to prostaglandin receptor EP1,
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a nuclear protein, which can increase gene expression leading to uncontrolled cell proliferation. We further investigated the effect of genistein on COX-2 expression and activity. Our results showed that genistein exerted no effect on COX-2 protein expression (Fig. 5). However, it directly inhibited COX-2 enzymatic activity, similar to the action of celecoxib, a COX-2 specific inhibitor (Fig. 4B). Compared to celecoxib it was less effective, indicating that genistein may bind to COX-2 at a different site from celecoxib or with weaker affinity [32]. In summary, genistein exerts some anticancer activity through the action of decreasing cell proliferation. This activity may be related to suppression of PCNA expression and reduction of PGE2 synthesis via inhibition of COX-2 activity. However, its anticancer activity is much weaker compared to other COX specific inhibitors and flavonoids isolated from other plants. Therefore, genistein may be more useful in chemoprevention rather than chemotherapy for HNSCC.
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