Essential Role of P53 in Phenethyl Isothiocyanate-Induced Apoptosis1

[CANCER RESEARCH 58. 4102-4106, September 15. 1998] Advances in Brief

Essential Role of p53 in Phenethyl Isothiocyanate-induced Apoptosis1

Chuanshu Huang, Wei-ya Ma, Jingxia Li, Stephen S. Hecht, and Zigang Dong2

The Hormel InstÃlale, University of Minnesota, Austin, Minnesota 55912 [C. H., W-y. M., J. L, Z. D.Â¡:and University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Â¡S.S. HJ

Abstract chemically induced lung, forestomach, and esophageal tumorigenesis (16-21, 23). It has been hypothesized that the inhibitory action of Phenethyl isothiocyanate (PEITC) is a natural product that is among PEITC is due to competitive inhibition and inactivation of enzymes, the most effective cancer chemopreventive agents known. Mechanistic such as cytochromes P450, involved in the bioactivation of carcino studies indicate that the chemopreventive activity of PEITC is associated gens (23-26). The chemopreventive properties of PEITC and other with its favorable modification of carcinogen metabolism and its induction of apoptosis. Here, we found that PEITC blocks tumor promoter (12-0- structurally related isothiocyanates are also associated with the induc tetradecanoylphorbol-13-acetate or epidermal growth factor)-induced cell tion of phase II detoxifying enzymes, including glutathione 5-trans- transformation in mouse epidermal JB6 cells, and this inhibitory activity ferases, quinone reducÃase,epoxide hydrolase, and UDP-glucurono- on cell transformation is correlated with induction of apoptosis. Most syltransferases (22, 23-26). Recently, it was reported that PEITC importantly, apoptosis induction by PEITC occurs through a p53-depend- induces activation of JNK activity and that this activation is associated ent pathway. This was demonstrated not only by results that PEITC with its apoptosis induction (27, 28). Here, we demonstrate that induction of p53 protein expression and p53-dependent transactivation PEITC also induces p53 transactivation in a dose- and time-dependent but also by PEITC-induced apoptosis inp53 +/+ cells but not inp53 -/- fashion and that this plays a crucial role in PEITC-induced apoptosis cells. In contrast, PEITC induced apoptosis in cells with both normal or deficient sphingomyelinase activity. Our results demonstrate for the first and its antitumor promotion effects in JB6 Cl 41 cells. time that p53 elevation is required for PEITC-induced apoptosis, which may be involved in its cancer chemopreventive activity. Materials and Methods

Introduction Reagents. PEITC was obtained from Aldrich Chemical Co. (Milwaukee, WI), and protein G plus protein A agarose, monoclonal mouse IgG against p53 p53 is one of the classical tumor suppressor genes that interferes (Abl). and polyclonal rabbit IgG against p53 (AblO) were from Oncogene with cell transformation events (1, 2). p53 also plays a critical role in Research Products. TPA was purchased from Sigma Chemical Co.: EGF was cell cycle control and induction of apoptosis (3, 4). It is elevated in from Collaborative Research; luciferase assay substrate was from Promega; response to genotoxic agents, such as ionizing radiation, UV light, or and FBS, Eagle's MEM, DMEM, RPMI 1640, and BME were from BioWhit- chemicals (5, 6). taker. Cell Culture. JB6 P+ (tumor promoter-sensitive) mouse epidermal cell Apoptosis, or programmed cell death, has been characterized as a line C1 41 and its stable PG13 luciferase reporter plasmiti transfects, C1 41 p53 fundamental cellular mechanism that occurs under a range of physi cells, were cultured in monolayers at 37Â°Cand 5% CO, using Eagle's MEM ological and pathological conditions (5-9). It plays an essential role as containing 5% FCS, 2 HIM L-glutamine, and 25 /Â¿g/mlgentamicin (29, 30). a protective mechanism against neoplastic development in the orga EBV-transformed normal human lymphoblast JY cells and Niemann-Pick nism by eliminating genetically damaged or excess cells that have disease MS1418 lymphoblasts (31) were maintained in a mixture of RPMI been induced improperly to divide by a mitotic stimulus (10, 11). 1640 and DMEM (1:1, v/v) containing 15% FBS, 2 mM L-glutamine, and 25 Growing evidence from both in vitro and in vivo studies demonstrates mg/ml gentamicin. Normal mouse embryo fibroblasts (p53 +/+) and p53- that suppression of apoptosis is involved in tumor promotion by deficient embryo fibroblasts (p53 â€”Iâ€”)were cultured in DMEM with 10% chemical agents. Inhibition of apoptosis by phÃ©nobarbital,peroxisome FBS, 2 mM L-glutamine, and 25 mg/ml gentamicin (32). proliferators, cyproterone acetate, and dichloroacetic acid may be Anchorage-independent Transformation Assay. JB6 Cl 41 cells involved in their effect on liver carcinogenesis (12-14). The tumor (1 X IO4) were exposed to TPA (10 ng/ml) or EGFflO ng/ml) with orwithout promoter TPA3 inhibited apoptosis induced in cultures of C3H- different concentrations of PEITC in 1 ml of 0.33% BME agar containing 10% 10T1/2 cells by exposure to ionizing radiation, ÃŸ-radiation,or acute FBS over 3.5 ml of 0.5% BME agar medium containing 10% FBS. The cultures were maintained in a 37Â°C/5%CO, incubator for 14-21 days, and the serum deprivation (15). Therefore, suppression of apoptosis may be a feature of tumor promotion by chemical carcinogens. colonies were scored by the methods described (29, 33). Assay for p53-dependent Transcription Activity. Confluent monolayers PEITC, which occurs as a conjugate in certain cruciferous vegeta of Cl 41 p53 cells were trypsinized, and 8 X IO3 viable cells suspended in 100 bles, has been extensively investigated for its chemopreventive activ ju.1of 5% FBS MEM were added to each well of a 96-well plate. Plates were ity against cancer in rats and mice (16-22). It has effectively inhibited incubated at 37Â°Cin a humidified atmosphere of 5% CO2. Twelve to 24 h later, cells were starved by culturing them in 0.1% FBS MEM for 12 h. The cells Received 6/5/98; accepted 7/30/98. were exposed to different concentrations of PEITC for p53 induction for 24 h. The costs of publication of this article were defrayed in part by the payment of page The cells were extracted with lysis buffer, and luciferase activity was measured charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. using a luminometer (Monolight 2010). The results are expressed as relative 1This research was supported by The Hormel Foundation, National Cancer Institute p53-dependent transcription activity (30). Grants CA74916 and CA46535. and a Grant-in-Aid from the Graduate School of the Immunoprecipitation Assay. The level of p53 protein after PEITC treat University of Minnesota. 2 To whom requests for reprints should be addressed, at The Hormel Institute. Uni ment was measured by Western blot for immunoprecipitation with specific versity of Minnesota. 801 16th Avenue NE. Austin. MN 55912. Phone: (507) 437-9640; antibodies against p53. Briefly, 80% confluent JB6 Cl 41 cells cultured in 5% Fax: (507)437-9606: E-mail: [email protected]. 'The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; PEITC. FBS MEM were treated with PEITC (10 /MM)for different times. The cells phenethyl isothiocyanate: JNK. c-Jun NHi-terminal kinase: EGF. epidermal growth were then lysed on ice for 1 h in the lysis buffer and spun at 14,000 rpm for factor; FBS. fetal bovine serum: BME. basal medium Eagle's; SMase. sphingomyelinase; 5 min. The lysates were immunoprecipitated using p53 antibodies (Abl) and NNK, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone. protein G plus protein A agarose. The beads were washed, and the p53 protein 4102

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. p53 IS INVOLVED IN APOPTOSIS INDUCED BY PEITC was selectively measured by Western immunoblotting using a chemilumines- PEITC cent detection system and specific p53 antibodies (Ab 10). DNA Fragmentation Assay. Cells were treated with different concentra tions of PEITC for 24 h. All of the cells were harvested by centrifugation and lysed with a lysis buffer [5 mM Tris-HCl (pH 8.0). 20 mM EDTA, and 0.5% s n Triton X-100) on ice for 45 min. Fragmented DNA in the supernatant of a a. Â« 14,000 rpm centrifugation (30 min at 4Â°C)was extracted once with phenol/ i 8 IT) CVÃŒ chloroform/isoamyl alcohol (25:24:1, v/v/v) and once with chloroform and kb precipitated by ethanol and salt. The DNA pellet was washed once with 70% ethanol and resuspended in Tris-EDTA buffer (pH 8.0) with 100 ng/ml RNase 12.0 at 37Â°Cfor 2 h. The DNA fragments were separated in 1.8% agarose gel 3.0 electrophoresis and visualized under UV light. 2.0 Results 1.6 Inhibition of TP A- and EGF-induced Cell Transformation by PEITC. We used the JB6 Cl 41 cell line, a well-characterized 1.0 late-stage tumor promotion cell culture model, to investigate the effects of PEITC. As shown in Fig. 1, PEITC inhibits tumor promoter (TPA or EGF)-induced cell transformation in a dose-dependent man 0.5 ner, ranging from 2.5 to 10 H.M(Fig. 1). Induction of Apoptosis in JB6 Cl 41 Cells by PEITC. Inhibition 0.3 of apoptosis is one of the mechanisms of tumor formation and chemopreventive agents may act through the induction of apoptosis to Fig. 2. Induction of apoptosis by PEITC in JB6 CI 41 cells. Subconfluem (80-90%) block the carcinogenesis process (14, 15, 34, 35). We, therefore, monolayer JB6 Cl 41 cells in 100-mm dishes were subjected to different concentrations of PEITC for 16 h. Then, both delached and attached cells were harvested for DNA hypothesized that the induction of apoptosis may be involved in the fragmentation assay as described in "Materials and Melhods." anticarcinogenic effect of PEITC. To test this, we treated JB6 Cl 41 cells with different concentrations of PEITC. The results from DNA fragmentation assays show that treatment of cells with PEITC induced duction by PEITC was also reported in HeLa cells and HT 1080 cells apoptosis in the Cl 41 cells in the same dose range at which it by others (27). inhibited cell transformation (Fig. 2). These results support our notion No Difference in Apoptosis Induction between a Cell Line with that the cancer-preventive effect of PEITC may occur partially Wild-Type SMase and a SMase-deficient Cell Line. A cell will through the induction of apoptosis. During this study, apoptosis in- undergo apoptosis as a result of information received from its envi ronment and interpreted in the context of internal information (36). Although the intracellular signals involved in induction of apoptosis 8400 are often involved in promotion of proliferation or differentiation in other cellular contexts, some pathways appear to be of particular significance in the control of cell apoptosis. It is known that ceramide and p53 are of particular importance in the induction of apoptosis in many instances (36-38). Ceramide and/or SMase activity is required for many agents such as tumor necrosis factor-a, IFN, or y-ionizing radiation to induce apoptosis (11-13, 31, 38). To assess the role of ceramide/SMase in PEITC-induced apoptosis, we used a SMase- deficient lymphoblast cell line, MS 1418, and a normal lymphoblast control cell line, JY (31). The results show that PEITC induced apoptosis in both cell lines in the same dose ranges (Fig. 3). These data rule out the involvement of ceramide/SMase in PEITC-induced apoptosis. Treatment of JB6 Cells with PEITC Results in p53 Transacti- vation and Expression. Normal p53 function is thought to be crucial in the induction of apoptosis in human and murine cells following DNA damage (39, 40). This notion was further supported by the findings that p53 is the most commonly mutated tumor suppressor gene and that lack of p53 expression or function is associated with an increased risk of tumor formation (41-43). Apoptosis of thymocytes and intestinal crypt cells following irradiation was almost completely blocked in p53-deficient mice (40), and incidence of spontaneous tumors in p53 â€”Iâ€”mice was high and occurred rapidly (44). To TPA (10 ng/ml) EGF (10 ng/ml) + investigate the possible role of p53 in the induction of apoptosis by PEITC (^M) 2.5 10 2.5 10 PEITC, we analyzed the influence of PEITC on p53-dependent tran scription activity and p53 protein expression in PG13-luciferase JB6 Fig. 1. Inhibition of EGF- or TPA-induced JB6 Cl 41 cell transformation by PEITC. A total of IO4 JB6 Cl 41 cells were exposed simultaneously lo either TPA (10 ng/ml) or cell transfectants (30). We found that PEITC markedly activates the EGF (10 ng/ml). with or without different concentrations of PEITC in 0.33% BME agar p53-dependent transcription activity in a dose-dependent manner (Fig. containing 10% FBS over 0.5% BME agar containing 10% FBS. Cell colonies were scored after 14 days of incubation at 37Â°Cin 5% CO2. The results are expressed as 4A). The dose ranges of PEITC for transactivating p53 activity are described previously (29. 33). Columns, means of values from triplicate assays: bars. SD. consistent with those for their induction of apoptosis and inhibition of 4103

JY MS1418 Chinese cabbage (16-23). Here, we investigated the effect of PEITC in a mouse epidermal tumor promotion cell culture model. The results show that PEITC blocks tumor promoter (EOF or TPA)-induced cell transformation and induces cell apoptosis at the same dose, ranging

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Pig. 3. PEITC induces apoptosis in both JY and MS1418 cells. SMase-deficient MS 1418 cells or normal lymphoblast JY cells were Irealed with PEITC for 12 h. Then, the i cells were harvested and DNA fragmentation assays were performed as described in "Materials and Methods." o â€¢eE. o cell transformation (Figs. 1, 2, and 4A). Maximum transactivation of co p53 activity was observed from 9 to 24 h after the cells were exposed â€¢4-Â«Â§ to PEITC (Fig. 4B). Treatment of cells with PEITC also led to an "E elevated level of p53 protein (Fig. 4C). These results indicate that "OO) induction of p53 protein by PEITC is at least partially responsible for glucosinolate in a levels of p53 protein were measured by Western blot using p53 antibody (AbIO; Onco variety of cruciferous vegetables, such as watercress, turnips, and gene Research Products) and a chemiluminescent detection system. 4104

P53+/+ P53-/- Apoptosis is a crucial element in the behavior of mammalian cells in many different situations. It is believed that apoptosis is responsible for the deletion of unwanted cells during organ and tissue develop ment and pathologically induced tissue damage as well as excess cells that have improperly been induced to divide by a mitotic stimulus (10, â€¢iin io 11). In addition, apoptosis of individual cells may represent a protec in cJ in evi tive mechanism against neoplastic development in the organism by eliminating genetically damaged cells (10, 11). A cell will undergo o 2000 EOOO â€¢Â£cbbbcbbb apoptosis as a result of signals received from its environment, inter CO OLULULUOLULULU preted in the context of internal signals. External signals trigger 2 UQ_Q_CLOQ_D.CL apoptosis through a signal transduction pathway, which may involve kb the stimulation of the receptor, the activation of protein kinase/ phosphatase cascades, and the release of second messengers to up- 12.0 regulate or suppress the transcription of specific genes (50). Although the great variety of external signals that can control apoptosis means 3.0 that many signaling pathways can be involved, p53 and ceramide are 2.0 the two most extensively investigated pathways (36-38). Ceramide 1.6 and/or SMase activity is required for many agents such as tumor necrosis factor-a, IFN, and -y-ionizing radiation to induce apoptosis 1.0 (10, 11, 38). Normal p53 function was shown to be crucial in the induction of apoptosis in human and murine cells following DNA 0.5 damage (50). This notion was supported by the findings that p53 mutations occur in 50-55% of all human cancers (50) and that these 0.3 mutations are strongly selected for p53 proteins that fail to bind to DNA in a sequence-specific fashion. Lack of p53 expression or function is associated with an increased risk of tumor formation Fig. 5. PEITC induces apoptosis in p53 +/+ but not in p53 -I- fibroblasts. (41-43). Transgenic mice expressing the SV40 large T antigen Subconfluent monolayers of p53 +/+ or p53 â€”Iâ€”cellsin 100-mm dishes were treated develop choroid plexus papillomas during the first 3 months of life with 10 JIM PEITC for 12 h. Then, both the detached and attached cells were harvested for DNA fragmentation assays as described in "Materials and Methods." (51). The tumor growth is slowed considerably by p53-mediated apoptosis when a T antigen mutant is used that cannot inactivate p53 function in these cells (51). When this mutant is expressed in from 2.5 to 10 JU.M.Although blood levels of PEITC have not been p53 -Iâ€” mice, the rapid tumor growth resumes with less apoptosis reported, consumption of watercress releases 10-40 mg of PEITC, (52). Apoptosis of intestinal crypt cells following irradiation was suggesting that concentrations in the micromolar range may be almost completely blocked in p53-deficient mice (40). Further achieved in vivo (45, 46). Because PEITC-induced apoptosis is asso more, incidence of spontaneous tumors in p53 â€”Iâ€”micewas high ciated with p53 transactivation and this apoptosis induction was only and occurred rapidly (44). Hence, p53-mediated apoptosis clearly observed in p53 +/+, not in p53 â€”Iâ€”cells, we suggest that p53 is an important part of the tumor suppressor phenotype and sub activation is required for PEITC-induced apoptosis and is involved in sequent p53 deficiency may permit a population of genetically the PEITC-associated chemoprevention of cancer. damaged cells to escape the normal process of apoptotic deletion. PEITC has attracted a great deal of attention because it inhibits Here, we found that PEITC markedly activates p53-dependent carcinogenesis in several animal models, including /V-nitrosometh- transcription activity in a dose-dependent manner. It was known ylbenzylamine-induced-esophageal tumors in rats, /V-nitroso-bis- that p53 was activated in response to DNA damage. The activation (2-oxopropyl)amine-induced pancreatic tumors in hamsters, and of p53 may be due to DNA damage and chromosomal aberrations NNK-induced lung tumorigenesis in rats (16-22, 46, 47). The caused by PEITC (53). We also demonstrated that PEITC induced mechanism of inhibition of NNK-induced carcinogenesis by p53 protein expression and transactivation in the same dose range PEITC has been proposed in previous studies. It is suggested that PEITC's effect on NNK carcinogenesis is due to inhibition of NNK as that which inhibited cell transformation and induced apoptosis metabolic activation to methylating and pyridyloxobutylating elec- in the same cell line. These results suggest that increased p53 protein and p53-dependent transcriptional activity may be associ trophiles and enhancement of its detoxification (24, 26, 46-48). ated with PEITC-induced apoptosis and antitumor activity of The persistent inhibition of NNK metabolic activation resulted from inhibition of cytochrome P-450 enzymes (24, 26, 47, 48). PEITC. The strongest evidence for the role of p53 in PEITC- Very recently, it was reported that PEITC could induce JNKs induced apoptosis was provided by the study using cell lines with wild-type p53 (p53 +/+) or p53 deficiency (p53 â€”/â€”).Apoptosis activation and apoptosis in HeLa cells (27, 28). Here, we demon strated that PEITC also inhibits TPA- or EGF-induced cell trans induction by PEITC was only observed in p53 +/+ cells, not in p53 -I- cells, whereas apoptosis was seen in both the SMase- formation and induces apoptosis at the same dose ranges in a mouse epidermal JB6 cell line, suggesting that apoptosis induction deficient cell line and the cell line with wild-type SMase. by PEITC may be involved in its antitumor activity. Considering In summary, we demonstrate here that PEITC induces apoptosis in that JNK increases phosphorylation of p53 at serine 34 (49) and the same concentration range at which it inhibits cell transformation. plays an important role in PEITC-induced apoptosis (27), we PEITC-induced apoptosis occurs through a p53-dependent, SMase- suggest that PEITC-induced apoptosis may be through JNK, independent pathway. We suggest that the induction of apoptosis p53 phosphorylation, and a p53-dependent transcriptional activity mediated by p53 is a possible explanation for the antitumor promotion pathway. effect of PEITC in the JB6 cell model. 4105

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Chuanshu Huang, Wei-ya Ma, Jingxia Li, et al.

Cancer Res 1998;58:4102-4106.

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