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Phenethyl Isothiocyanate-induced Apoptosis in p53-deficient PC-3 Human Prostate Cancer Cell Line Is Mediated by Extracellular Signal-regulated Kinases¹

Department of Pharmacology and the University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Previous studies have suggested that p53 is required for apoptosis induction by phenethyl isothiocyanate (PEITC), which is a highly promising cancer chemopreventive agent. Here, we report that p53 is not required for PEITC-induced apoptosis in the PC-3 human prostate cancer cell line and that the PEITC-induced apoptosis is mediated by extracellular signal-regulated kinases (ERK1/2). Exposure of PC-3 cells to an apoptosis-inducing concentration of PEITC (10 µM) resulted in a rapid and sustained activation of ERK1/2 that was evident as early as 1 h after PEITC treatment and persisted for the duration of the experiment (24-h after PEITC exposure). The PEITC-mediated activation of ERK1/2 was associated with an increase in phosphorylation of its substrate Elk-1 at Ser383. The PEITC-induced activation of ERK1/2 as well as apoptosis was abolished in the presence of mitogen-activated protein/ERK kinase 1 (a kinase upstream of ERK1/2) inhibitor PD98059. Exposure of PC-3 cells to 10 µM PEITC also resulted in a time-dependent activation of p38 protein kinase that was associated with increased phosphorylation of activating transcription factor 2 at Thr71. Even though the PEITC-induced activation of p38 protein kinase was abrogated in the presence of its specific inhibitor SB202190, inhibition of p38 protein kinase activation did not prevent PEITC-induced apoptosis. In contrast to previous reports in other cellular systems, c-Jun NH₂-terminal kinases were not activated by PEITC treatment in PC-3 human prostate carcinoma cell line. In conclusion, the results of the present study indicate that p53 is not essential for PEITC-induced apoptosis and that the PEITC-induced apoptosis in PC-3 human prostate carcinoma cell line is mediated by ERKs. Thus, it seems reasonable to postulate that PEITC may be effective against tumors with normal as well as mutant p53.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Dietary ITCs,³ such as PEITC, are present in rather large quantities in cruciferous vegetables including broccoli, cabbage, watercress, and so forth (1) . ITCs are generated through myrosinase-catalyzed hydrolysis of corresponding glucosinolates (reviewed in Refs. 1 , 2 ). Human epidemiological studies indicate a statistically significant inverse correlation between dietary intake of ITC-containing vegetables and the risk for prostate cancer (3 , 4) . The evidence for cancer-protective effect of ITCs also derives from animal studies (5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . Chemopreventive activity for naturally occurring ITCs and/or their thiol conjugates has been demonstrated against benzo(a)pyrene-induced pulmonary and forestomach cancers in mice (5) , N-nitrosobenzylmethylamine-induced esophageal tumorigenesis in rats (6 , 7) , 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary neoplasia in mice (8, 9, 10) , and azoxymethane-induced colonic aberrant crypt foci in rats (12) . Mechanistic studies have indicated that chemopreventive activity of ITCs against chemically induced cancers is because of their ability to increase detoxification of the activated carcinogenic metabolites through induction of Phase II drug metabolizing enzymes, including glutathione transferases and quinone reductase (15 , 16) . Certain ITCs are believed to exert anticancer activity by reducing carcinogen activation via inhibition of cytochrome P450-dependent monooxygenases (15 , 16) .

Recent studies have indicated that certain ITCs inhibit growth of cancer cells in culture by inducing apoptosis. For example, PEITC is shown to induce apoptotic cell death in HeLa cells in a time- and concentration- dependent manner (17) . The PEITC-induced apoptosis was shown to be associated with a rapid and transient induction of caspase-3-like activity and activation of JNKs (17 , 18) . The involvement of caspases, including caspase-3 and caspase-8, and JNKs in PEITC-induced apoptosis has also been established in other cell lines including human leukemia HL-60 cells (19 , 20) . In addition, Huang et al. (21) have demonstrated that PEITC effectively blocks tumor promoter (12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor)-induced cell transformation in mouse epidermal JB6 cells by inducing apoptosis. The PEITC-induced apoptosis in JB6 cells was associated with transactivation and increased expression of p53 (21) . These investigators also showed that embryonic fibroblasts from p53-deficient mice are resistant to PEITC-induced apoptosis (21) . On the basis of these novel findings, it was hypothesized that p53 is essential for PEITC-induced apoptosis (21) . The present studies were designed to determine whether p53 dependence of PEITC-induced apoptosis is unique to embryonic fibroblasts. The results of the present study clearly indicate that PEITC is a highly potent inducer of apoptosis in a p53-deficient PC-3 human prostate cancer cell line and that PEITC-induced apoptosis is mediated by ERKs.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Reagents.
PEITC was purchased from Aldrich (Milwaukee, WI). F-12K Nutrient Mixture (Kaighn’s modification), PSN antibiotic mixture, and nonheat inactivated fetal bovine serum were from Life Technologies, Inc. (Grand Island, NY); sulforhodamine B and propidium iodide were from Sigma (St. Louis, MO); reagents for electrophoresis were from Bio-Rad (Richmond, CA); ApoAlert Annexin V-FITC apoptosis kit was from Clontech (Palo Alto, CA); and p44/42 (ERK1/2) and p38 MAPK assay kits were from Cell Signaling Technology (Beverly, MA). The MAPK inhibitors PD98059 and SB202190 were purchased from Calbiochem Technology (San Diego, CA). Antibodies against ERK1/2 were from BD Biosciences (San Diego, CA), whereas antibodies against p53, phospho-ERK1/2, p38, and phospho-p38 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture.
Monolayer cultures of PC-3 cells, which were generously provided by Dr. Candace Johnson (University of Pittsburgh, Pittsburgh, PA), were maintained in F-12K Nutrient Mixture supplemented with 7% (v/v) non-heat inactivated fetal bovine serum and PSN antibiotic mixture in a humidified atmosphere of 5% CO₂ and 95% air at 37°C.

Sulforhodamine B Assay.
Effect of PEITC on survival of PC-3 cells was determined by a modified sulforhodamine B assay (22) . Briefly, cells (2 x 10³ cells/well) were plated in 96-well microtiter plates and allowed to attach overnight. Medium was replaced with fresh complete medium containing desired concentrations of PEITC. Stock solution of PEITC was prepared in DMSO, and an equal volume of DMSO (final concentration <1%) was added to control wells. After a 24-h incubation at 37°C, a 10% (w/v) aqueous (100 µl) solution of ice-cold trichloroacetic acid was added to each well, and the incubation was continued for 1 h. Plates were washed with deionized water, allowed to air dry, and stained with 100 µl of 0.4% sulforhodamine B solution in 1% acetic acid for 15 min. Subsequently, plates were washed five times with 1% acetic acid to remove unbound dye, and the plates were allowed to air dry. A solution containing 10 mmol/liter Tris base (pH 10.5; 150 µl) was then added to each well, and the absorbance was measured at 570 nm using a Labsystems Multiskan plus plate reader.

Determination of Apoptosis.
Apoptotic cells were quantified using ApoAlert Annexin V-FITC kit according to the manufacturer’s instructions. Briefly, PC-3 cells (10⁶) were exposed to desired concentration of PEITC in the absence or presence of PD98059 or SB202190 for 24 h at 37°C. Subsequently, both floating and adherent cells were collected. The floating cells were collected by centrifugation at 700 x g for 5 min, whereas adherent cells were harvested by trypsinization and collected by centrifugation at 700 x g for 5 min. Pooled cells were washed with the manufacturer-supplied 1 x binding buffer. Approximately 5 x 10⁵ cells were resuspended in 200 µl of manufacturer-supplied 1 x binding buffer, and mixed with 5 µl of Annexin V-FITC and 10 µl of propidium iodide. After 15 min of incubation in the dark, the cells were analyzed using a Coulter Epics XL flow cytometer.

Western Blot Analysis, and p44/42 and p38 MAPK Assays.
PC-3 cells were treated with desired concentration of PEITC in the absence or presence of MAPK inhibitors for specified time intervals. For Western blotting, the cells were washed twice with ice-cold PBS, and lysed on ice for 40 min in a solution containing 50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM Na₃VO₄, 2 mM EGTA, 12 mM ß-glycerolphosphate, 10 mM NaF, 16 µg/ml benzamidine hydrochloride, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was centrifuged at 14,000 x g for 15 min, and the supernatant fraction was collected for Western blotting. Protein content in the supernatant fraction was determined by the method of Bradford (23) . Supernatant fraction proteins were separated by SDS-PAGE according to the method of Laemmli (24) . The proteins were transferred onto polyvinylidene fluoride membrane as described by Towbin et al. (25) . After blocking for 1 h in 10% nonfat dry milk in Tris-buffered saline, the membrane was incubated with desired primary antibody for 1 h. The membrane was then treated with appropriate peroxidase-conjugated secondary antibody, and the immunoreactive proteins were detected using enhanced chemiluminescence kit (NEN Life Science Products, Boston, MA) according to the manufacturer’s instructions. Each membrane was stripped and reprobed with antibodies against actin to correct for differences in protein loading. The p44/42 (ERK1/2) MAPK and p38 MAPK activities were determined using kits from Cell Signaling Technology (Beverly, MA) according to the manufacturer’s instructions.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
As shown in Fig. 1A , survival of PC-3 cells was reduced significantly by a 24-h exposure to PEITC in a concentration-dependent manner with an IC₅₀ of 6 µM. To determine whether reduced survival of PC-3 cells in the presence of PEITC was associated with apoptosis induction, apoptotic cells were quantified in cultures of control (DMSO-treated) and PEITC-treated PC-3 cells using FITC-conjugated Annexin V kit. Annexin V is a calcium-dependent phospholipid binding protein with a high affinity for phosphatidylserine, which is a negatively charged membrane phospholipid located on the inner (cytoplasmic) surface of the plasma membrane of living cells (26) . In cells undergoing apoptosis, phosphatidylserine is translocated to the outer side of the plasma membrane and becomes accessible for staining with Annexin V (27) . Staining with propidium iodide, a nucleic acid binding dye that is impermeable in live cells, is indicative of necrosis. Fig. 1B depicts the percentage of apoptotic cells after a 24-h treatment of PC-3 cells to increasing concentrations of PEITC (2.5, 5, and 10 µM). As can be seen in Fig. 1B , PEITC treatment resulted in an increase in percentage of apoptotic cells in a concentration-dependent manner. In comparison with DMSO-treated control PC-3 cells where roughly 4% of the cells were apoptotic, the percentage of apoptotic cells was increased by about 1.8–7.9-fold on PEITC treatment. Previous studies have shown that the PC-3 cell line lacks normal p53 expression (28 , 29) , which was confirmed in the present study through Western blot analysis of PC-3 cell lysate using antibodies against p53 (data not shown). These results clearly indicated that p53 is not required for PEITC-induced apoptosis in PC-3 human prostate cancer cell line.

View larger version (21K): [in this window] [in a new window]   Fig. 1. A, survival of PC-3 human prostate cancer cell line after a 24-h exposure to different concentrations of PEITC. Cell survival was determined by a modified sulforhodamine B assay as described in "Materials and Methods." Results are expressed as percentage of vehicle (DMSO) -treated control cells, and are mean of at least three determinations. B, effect of PEITC treatment on apoptosis induction in PC-3 cells. The PC-3 cells were exposed to either DMSO (control) or different concentrations of PEITC for 24 h. Both floating and adherent cells were collected, and approximately 5 x 105 cells were stained with Annexin V-FITC and propidium iodide as described in "Materials and Methods." After 15 min of incubation, the cells were analyzed using a Coulter Epics XL flow cytometer. Data are mean of two to three experiments; bars, ± SD.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Effect of PEITC treatment on activation and expression (A and B), and kinase activity of ERKs (C) in PC-3 cells. For data in A, the cells were treated with 10 µM PEITC for different time points. The control cells received an equal volume of DMSO. The cell lysates were prepared, and Western blotting was performed using antibodies against ERK1/2 and phospho-ERK1/2. B summarizes the PEITC-mediated activation of ERK1/2 relative to control after correction for differences in protein loading (reprobing the blots with antiactin antibodies). Data are mean of two determinations. C shows the effect of PEITC treatment on kinase activity of ERKs as measured by phosphorylation of Elk-1 at Ser383. The kinase activity was determined using a kit from Cell Signaling Technology (Beverly, MA) according to the manufacturer’s instructions. Comparable data were obtained in duplicate determinations; bars, ± SD.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Effect of treatment of PC-3 cells with the MEK1 inhibitor PD98059 on PEITC-mediated activation of ERK1/2 (A and B) and PEITC-induced apoptosis (C). A, Western blot analysis using lysates from cells exposed to 10 µM PEITC for 24 h either in the absence (Lane 3) or presence of 50 µM PD98059 (Lane 4). Lanes 1 and 2, respectively, contained lysates from cells treated with DMSO (control) and PD98059 alone. The blot was stripped and reprobed with antiactin antibodies to correct for differences in protein loading. B summarizes the effect of PD98059 on PEITC-mediated activation of ERK1/2. Data are means of two experiments. C shows the effect of PD98059 on PEITC-induced apoptosis. The cells were treated with 5 or 10 µM PEITC for 24 h either in the absence or presence of 50 µM PD98059. An equal volume of DMSO was added to the control cultures. Apoptotic cells were quantified by flow cytometric analysis after staining with Annexin V-FITC. Data are means (n = 3); bars, ± SD.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Effect of PEITC treatment on activation and expression (A and B) and kinase activity of p38 protein kinase (C) in PC-3 cells. A, cells were treated with 10 µM PEITC for different time points. The control cells received an equal volume of DMSO. The cell lysates were prepared, and Western blotting was performed using antibodies against p38 and phospho-p38. B summarizes time-dependent activation of p38 protein kinase relative to control after correction for differences in protein loading. Data are mean of two determinations. C shows the effect of PEITC treatment on kinase activity of p38 as measured by phosphorylation of ATF-2 at Thr 71. The kinase activity was determined using a kit from Cell Signaling Technology (Beverly, MA) according to the manufacturer’s instructions; bars, ± SD.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effect of p38 protein kinase inhibitor SB202190 on PEITC-mediated activation of p38 protein kinase (A and B) and PEITC-induced apoptosis (C). A, Western blot analysis using lysates from cells exposed to 10 µM PEITC for 24 h either in the absence (Lane 3) or presence of 25 µM SB202190 (Lane 4). Lanes 1 and 2 contained lysates from cells treated with DMSO (control) and SB202190 alone, respectively. The blot was stripped and reprobed with anti-actin antibodies to correct for differences in protein loading. B summarizes the effect of SB202190 on PEITC-mediated activation of p38 protein kinase. Data are means of two to three experiments. C shows the effect of SB202190 on PEITC-induced apoptosis. The cells were treated with 5 or 10 µM PEITC for 24 h either in the absence or presence of 25 µM SB202190. An equal volume of DMSO was added to the control cultures. Apoptotic cells were quantified by flow cytometric analysis after staining with Annexin V-FITC. Data are means (n = 3); bars, ± SD.

The second goal of the present study was to elucidate the signal transduction mechanisms responsible for apoptosis induction by PEITC. Previous studies have shown that PEITC-induced apoptosis in HeLa, Jurkat, and 293 cells is associated with activation of JNKs (18) . PEITC-mediated activation of JNKs has also been observed in human leukemia HL-60 cell line (20) . The results of the present study indicate that PEITC-induced apoptosis in the PC-3 cell line is independent of JNK activation (data not shown). In mammalian cells, two other Ser/Thr MAP kinase cascades, ERKs and p38 protein kinase, are functional that are implicated in regulation of apoptosis in response to different stimuli (30) . We found that the exposure of PC-3 cells to an apoptosis-inducing concentration of PEITC results in a rapid and sustained activation of both ERK1/2 and p38 protein kinases in a time-dependent manner. The activation of ERK1/2 was evident as early as 1 h after PEITC treatment, whereas maximal activation of p38 kinase was not observed until the 4-h time point. The PEITC-induced ERK1/2 activation as well as apoptosis was completely abolished by an inhibitor of MEK1, which is an upstream kinase in ERK1/2-signaling cascade. Interestingly, inhibition of p38 kinase did not affect PEITC-induced apoptosis even though the PEITC-mediated activation of p38 kinase was significantly inhibited. These results indicate that PEITC-induced apoptosis in PC-3 cells is mediated by ERKs but independent of p38 protein kinase. In a recent study, inhibition of benzo(a)pyreneinduced lung tumorigenesis in A/J mice by dietary N-acetyl cysteine conjugate of PEITC during the postinitiation phase was shown to be associated with activation of all three of the MAPKs (14) . Thus, it seems reasonable to conclude that activation of MAPKs is an important event in chemopreventive activity of ITCs. However, the mechanism by which PEITC activates MAPKs remains to be established.

In conclusion, the present study demonstrates that: (a) PC-3 prostate cancer cell line is highly sensitive to growth inhibition by PEITC; (b) reduced survival of PC-3 cells after exposure to PEITC is associated with apoptosis induction; (c) PEITC can induce apoptosis in absence of normal p53; and (d) PEITC-induced apoptosis in the PC-3 cell line is mediated by activation of ERKs but not JNKs or p38 kinase.

	ACKNOWLEDGMENTS

 
We thank Karen Lew for technical assistance and preparation of the figures.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by USPHS grants CA55589 and CA91478, awarded by the National Cancer Institute.

² To whom requests for reprints should be addressed, at S-871 Scaife Hall, University of Pittsburgh, 3550 Terrace Street, Pittsburgh, PA 15261. Phone: (412) 648-3769; Fax: (412) 383-9822; E-mail: singhs{at}msx.upmc.edu

³ The abbreviations used are: ITC, isothiocyanate; PEITC, phenethyl isothiocyanate; JNK, c-Jun NH₂-terminal kinase; ERK, extracellular signal regulated kinase; MEK, mitogen-activated protein/ERK kinase; MAPK, mitogen-activated protein kinase.

Received 5/ 2/02. Accepted 5/17/02.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Fahey J. W., Zalcmann A. T., Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry (Oxf.), 56: 5-51, 2001.[Medline]
2. Zhang Y., Talalay P. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Res., 54: 1976s-1981s, 1994.[Medline]
3. Cohen J. H., Kristal A. R., Stanford J. L. Fruit and vegetable intakes and prostate cancer risk. J. Natl. Cancer Inst., 92: 61-68, 2000.[Abstract/Free Full Text]
4. Kolonel L. N., Hankin J. H., Whittemore A. S., Wu A. H., Gallagher R. P., Wilkens L. R., John E. M., Howe G. R., Dreon D. M., West D. W., Paffenbarger R. S., Jr. Vegetables, fruits, legumes and prostate cancer: a multiethnic case-control study. Cancer Epidemiol. Biomark. Prev., 9: 795-804, 2000.[Abstract/Free Full Text]
5. Wattenberg L. W. Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine or benzo(a)pyrene on pulmonary and forestomach neoplasia in A/J mice. Carcinogenesis (Lond.), 8: 1971-1973, 1987.[Abstract/Free Full Text]
6. Stoner G. D., Morrissey D. T., Heur Y-H., Daniel E. M., Galati A. J., Wagner S. A. Inhibitory effects of phenethyl isothiocyanate on N-nitrosobenzylmethylamine carcinogenesis in the rat esophagus. Cancer Res., 51: 2063-2068, 1991.[Abstract/Free Full Text]
7. Morse M. A., Zu H., Galati A. J., Schmidt C. J., Stoner G. D. Dose-related inhibition by dietary phenylethyl isothiocyanate of esophageal tumorigenesis and DNA methylation induced by N-nitrosomethylbenzylamine in rats. Cancer Lett., 72: 103-110, 1993.[Medline]
8. Morse M. A., Amin S. G., Hecht S. S., Chung F-L. Effects of aromatic isothiocyanates on tumorigenicity. O⁶-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung. Cancer Res., 49: 2894-2897, 1989.[Abstract/Free Full Text]
9. Jiao D., Eklind K. I., Choi C-I., Desai D. H., Amin S. G., Chung F-L. Structure-activity relationships of isothiocyanates as mechanism-based inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice. Cancer Res., 54: 4327-4333, 1994.[Abstract/Free Full Text]
10. Chung F-L., Morse M. A., Eklind K. I. New potential chemopreventive agents for lung carcinogenesis of tobacco-specific nitrosamines. Cancer Res., 52: 2719s-2722s, 1992.
11. Hecht S. S. Chemoprevention of lung cancer by isothiocyanates. Adv. Exp. Med. Biol., 401: 1-11, 1996.[Medline]
12. Chung F-L., Conaway C. C., Rao C. V., Reddy B. S. Chemoprevention of colonic aberrant crypt foci in Fisher rats by sulforaphane and phenethyl isothiocyanate. Carcinogenesis (Lond.), 21: 2287-2291, 2000.[Abstract/Free Full Text]
13. Hecht S. S. Inhibition of carcinogenesis by isothiocyanates. Drug Metab. Rev., 32: 395-411, 2000.[Medline]
14. Yang Y-M., Conaway C. C., Chiao J. W., Wang C-X., Amin S., Whysner J., Dai W., Reinhardt J., Chung F-L. Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and phenethyl isothiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis. Cancer Res., 62: 2-7, 2002.[Abstract/Free Full Text]
15. Talalay P., Fahey J. W. Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. J. Nutr., 131(Suppl.): 3027s-3033s, 2001.
16. Hecht S. S. Chemoprevention by isothiocyanates. J. Cell Biochem., 22(Suppl.): 195s-209s, 1995.
17. Yu R., Mandlekar S., Harvey K. J., Ucker D. S., Kong A. N. Chemopreventive isothiocyanates induce apoptosis and caspase-3-like protease activity. Cancer Res., 58: 402-408, 1998.[Abstract/Free Full Text]
18. Chen Y-R., Wang W. F., Kong A-N. T., Tan T. H. Molecular mechanisms of c-Jun N-terminal kinase-mediated apoptosis induced by anticarcinogenic isothiocyanates. J. Biol. Chem., 273: 1769-1775, 1998.[Abstract/Free Full Text]
19. Xu K., Thornalley P. J. Studies on the mechanism of the inhibition of human leukemia cell growth by dietary isothiocyanates and their cysteine adducts in vitro. Biochem. Pharmacol., 60: 221-231, 2000.[Medline]
20. Xu K., Thornalley P. J. Signal transduction activated by the cancer chemopreventive isothiocyanates: cleavage of BID protein, tyrosine phosphorylation and activation of JNK. Br. J. Cancer, 84: 670-673, 2001.[Medline]
21. Huang C., Ma W-Y., Li J., Hecht S. S., Dong Z. Essential role of p53 in phenethyl isothiocyanate-induced apoptosis. Cancer Res., 58: 4102-4106, 1998.[Abstract/Free Full Text]
22. Xiao D., Tan W. F., Li M. H., Ding J. Antiangiogenic potential of 10-hydroxycamptothecin. Life Sci., 69: 1619-1628, 2001.[Medline]
23. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976.[Medline]
24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.[Medline]
25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354, 1979.[Abstract/Free Full Text]
26. Fadok V. A., Voelker D. R., Campbell P. A., Bratton D. L., Cohen J. J., Henson P. M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol., 148: 2207-2216, 1992.[Abstract]
27. Martin S. J., Reutelingsperger C. P. M., McGahon A. J., Rader J. A., van Schie R. C. A. A., LaFace D. M., Green D. R. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med., 182: 1545-1556, 1995.[Abstract/Free Full Text]
28. Rokhlin O. W., Bishop G. A., Hostager B. S., Waldschmidt T. J., Sidorenko S. P., Pavloff N., Kiefer M. C., Umansky S. R., Glover R. A., Cohen M. B. Fas-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res., 57: 1758-1768, 1997.[Abstract/Free Full Text]
29. Whang Y. E., Wu X., Suzuki H., Reiter R. E., Tran C., Vessella R. L., Said J. W., Isaacs W. B., Sawyers C. L. Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression. Proc. Natl. Acad. Sci. USA, 95: 5246-5250, 1998.[Abstract/Free Full Text]
30. Xia Z., Dickens M., Raingeaud J., Davis R. J., Greenberg M. E. Opposing effects of ERK and JNK-p38 MAP kinases and apoptosis. Science (Washington, DC), 270: 1326-1331, 1995.[Abstract/Free Full Text]
31. Malkin D. The role of p53 in human cancer. J. Neuro-Oncol., 51: 231-243, 2001.[Medline]

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				C. Xu, X. Yuan, Z. Pan, G. Shen, J.-H. Kim, S. Yu, T. O. Khor, W. Li, J. Ma, and A.-N. T. Kong Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2. Mol. Cancer Ther., August 1, 2006; 5(8): 1918 - 1926. [Abstract] [Full Text] [PDF]

				J.-H. Kim, C. Xu, Y.-S. Keum, B. Reddy, A. Conney, and A.-N. T. Kong Inhibition of EGFR signaling in human prostate cancer PC-3 cells by combination treatment with {beta}-phenylethyl isothiocyanate and curcumin Carcinogenesis, March 1, 2006; 27(3): 475 - 482. [Abstract] [Full Text] [PDF]

				C. Xu, G. Shen, X. Yuan, J.-h. Kim, A. Gopalkrishnan, Y.-S. Keum, S. Nair, and A.-N. T. Kong ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and cell death elicited by three isothiocyanates in human prostate cancer PC-3 cells Carcinogenesis, March 1, 2006; 27(3): 437 - 445. [Abstract] [Full Text] [PDF]

				T. O. Khor, Y.-S. Keum, W. Lin, J.-H. Kim, R. Hu, G. Shen, C. Xu, A. Gopalakrishnan, B. Reddy, X. Zheng, et al. Combined Inhibitory Effects of Curcumin and Phenethyl Isothiocyanate on the Growth of Human PC-3 Prostate Xenografts in Immunodeficient Mice Cancer Res., January 15, 2006; 66(2): 613 - 621. [Abstract] [Full Text] [PDF]

				C. C. Conaway, C.-X. Wang, B. Pittman, Y.-M. Yang, J. E. Schwartz, D. Tian, E. J. McIntee, S. S. Hecht, and F.-L. Chung Phenethyl Isothiocyanate and Sulforaphane and their N-Acetylcysteine Conjugates Inhibit Malignant Progression of Lung Adenomas Induced by Tobacco Carcinogens in A/J Mice Cancer Res., September 15, 2005; 65(18): 8548 - 8557. [Abstract] [Full Text] [PDF]

				S. Dhanalakshmi, C. Agarwal, R. P. Singh, and R. Agarwal Silibinin Up-regulates DNA-Protein Kinase-dependent p53 Activation to Enhance UVB-induced Apoptosis in Mouse Epithelial JB6 Cells J. Biol. Chem., May 27, 2005; 280(21): 20375 - 20383. [Abstract] [Full Text] [PDF]

				M. E. Winters, A. I. Mehta, E. F. Petricoin III, E. C. Kohn, and L. A. Liotta Supra-additive Growth Inhibition by a Celecoxib Analogue and Carboxyamido-triazole Is Primarily Mediated through Apoptosis Cancer Res., May 1, 2005; 65(9): 3853 - 3860. [Abstract] [Full Text] [PDF]

				D. Xiao, Y. Zeng, S. Choi, K. L. Lew, J. B. Nelson, and S. V. Singh Caspase-Dependent Apoptosis Induction by Phenethyl Isothiocyanate, a Cruciferous Vegetable-Derived Cancer Chemopreventive Agent, Is Mediated by Bak and Bax Clin. Cancer Res., April 1, 2005; 11(7): 2670 - 2679. [Abstract] [Full Text] [PDF]

				H. K. KOUL, P. D. MARONI, R. B. MEACHAM, D. CRAWFORD, and S. KOUL p42/p44 Mitogen-Activated Protein Kinase Signal Transduction Pathway: A Novel Target for the Treatment of Hormone-Resistant Prostate Cancer? Ann. N.Y. Acad. Sci., December 1, 2004; 1030(1): 243 - 252. [Abstract] [Full Text] [PDF]

				J. M. Visanji, S. J. Duthie, L. Pirie, D. G. Thompson, and P. J. Padfield Dietary Isothiocyanates Inhibit Caco-2 Cell Proliferation and Induce G2/M Phase Cell Cycle Arrest, DNA Damage, and G2/M Checkpoint Activation J. Nutr., November 1, 2004; 134(11): 3121 - 3126. [Abstract] [Full Text] [PDF]

				N.-A. Pham, J. W. Jacobberger, A. D. Schimmer, P. Cao, M. Gronda, and D. W. Hedley The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice Mol. Cancer Ther., October 1, 2004; 3(10): 1239 - 1248. [Abstract] [Full Text] [PDF]

				S. K. Srivastava and S. V. Singh Cell cycle arrest, apoptosis induction and inhibition of nuclear factor kappa B activation in anti-proliferative activity of benzyl isothiocyanate against human pancreatic cancer cells Carcinogenesis, September 1, 2004; 25(9): 1701 - 1709. [Abstract] [Full Text] [PDF]

				B. Relic, V. Benoit, N. Franchimont, C. Ribbens, M.-J. Kaiser, P. Gillet, M.-P. Merville, V. Bours, and M. G. Malaise 15-Deoxy-{Delta}12,14-prostaglandin J2 Inhibits Bay 11-7085-induced Sustained Extracellular Signal-regulated Kinase Phosphorylation and Apoptosis in Human Articular Chondrocytes and Synovial Fibroblasts J. Biol. Chem., May 21, 2004; 279(21): 22399 - 22403. [Abstract] [Full Text] [PDF]

				D. Xiao, C. S. Johnson, D. L. Trump, and S. V. Singh Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells Mol. Cancer Ther., May 1, 2004; 3(5): 567 - 576. [Abstract] [Full Text] [PDF]

				J. M. Pullar, S. J. Thomson, M. J. King, C. I. Turnbull, R. G. Midwinter, and M. B. Hampton The chemopreventive agent phenethyl isothiocyanate sensitizes cells to Fas-mediated apoptosis Carcinogenesis, May 1, 2004; 25(5): 765 - 772. [Abstract] [Full Text] [PDF]

				N. Miyoshi, K. Uchida, T. Osawa, and Y. Nakamura A Link between Benzyl Isothiocyanate-Induced Cell Cycle Arrest and Apoptosis: Involvement of Mitogen-Activated Protein Kinases in the Bcl-2 Phosphorylation Cancer Res., March 15, 2004; 64(6): 2134 - 2142. [Abstract] [Full Text] [PDF]

				S. R. Whittaker, M. I. Walton, M. D. Garrett, and P. Workman The Cyclin-dependent Kinase Inhibitor CYC202 (R-Roscovitine) Inhibits Retinoblastoma Protein Phosphorylation, Causes Loss of Cyclin D1, and Activates the Mitogen-activated Protein Kinase Pathway Cancer Res., January 1, 2004; 64(1): 262 - 272. [Abstract] [Full Text] [PDF]

				A. V. Singh, D. Xiao, K. L. Lew, R. Dhir, and S. V. Singh Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo Carcinogenesis, January 1, 2004; 25(1): 83 - 90. [Abstract] [Full Text] [PDF]

				S. K. Srivastava, D. Xiao, K. L. Lew, P. Hershberger, D. M. Kokkinakis, C. S. Johnson, D. L. Trump, and S. V. Singh Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo Carcinogenesis, October 1, 2003; 24(10): 1665 - 1670. [Abstract] [Full Text] [PDF]

				V. W.Y. Lui, A. L. Wentzel, D. Xiao, K. L. Lew, S. V. Singh, and J. R. Grandis Requirement of a carbon spacer in benzyl isothiocyanate-mediated cytotoxicity and MAPK activation in head and neck squamous cell carcinoma Carcinogenesis, October 1, 2003; 24(10): 1705 - 1712. [Abstract] [Full Text] [PDF]