Sulforaphane Inhibits Constitutive and Interleukin-6–Induced Activation of Signal Transducer and Activator of Transcription 3 in Prostate Cancer Cells

Epidemiologic studies suggest that dietary intake of cruciferous vegetables may lower the risk of different malignancies, including prostate cancer (1–3). Antineoplastic activity of cruciferous vegetables is attributed to isothiocyanates, which are released by myrosinase-mediated hydrolysis of corresponding glucosinolates (4). Broccoli is a rich source of (−)-1-isothiocyanato-(4R)-(methylsulfinyl)-butane (L-SFN). The L-SFN and its synthetic derivative d,l-sulforaphane (SFN) have attracted a great deal of research inquiry because of their anticancer effects. For example, the L-SFN and SFN are equipotent inducers of quinone reductase activity in Hepa 1c1c7 hepatoma cells (5). The L-SFN or synthetic SFN has been shown to prevent chemically induced cancer in experimental animals, including 9,10-dimethyl-1,2-benzanthracene–induced mammary cancer in rats (6), azoxymethane-induced colonic aberrant crypt foci in rats (7), and benzo[a]pyrene-induced forestomach cancer in mice (8). Dietary feeding of SFN and its N-acetylcysteine conjugate inhibited malignant progression of lung adenomas induced by tobacco carcinogens in A/J mice (9).

In addition to prevention of chemically induced cancer in experimental animals, SFN treatment inhibits growth of human cancer cells in culture and in vivo (10–12). For example, growth of PC-3 human prostate cancer xenografts in athymic mice was inhibited significantly by oral or dietary administration of SFN (13, 14). Oral administration of SFN inhibited incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated prostate cancer as well as pulmonary metastasis multiplicity in a transgenic mouse model in association with augmentation of natural killer cell activity (15). Mechanistic knowledge explaining anticancer effect of SFN continues to expand, but the SFN-mediated suppression of cancer cell proliferation correlates with G₂-M–phase cell cycle arrest (10, 11, 16), apoptosis induction (10, 12, 17), inhibition of histone deacetylase (18), and protein binding (19). Mechanistic details of some of these cellular responses have been well described. For example, the SFN-induced G₂-M–phase cell cycle arrest in human prostate cancer cells is caused by checkpoint kinase 2–mediated Ser²¹⁶ phosphorylation of cell division cycle 25C (16). We also found that the SFN-induced apoptosis is selective toward cancer cells and intimately linked to production of reactive oxygen species (17, 20, 21). The SFN has been shown to inhibit activity of mitochondrial respiratory chain enzymes, leading to production of reactive oxygen species (21). The SFN-mediated suppression of androgen receptor signaling in prostate cancer cells has also been shown recently (22).

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor implicated in development and progression of various malignancies, including prostate cancer (23). STAT3 is constitutively activated in a variety of cancers, including breast and prostate cancers, melanoma, multiple myeloma, and leukemia (23–25). The present study shows that SFN treatment inhibits constitutive and interleukin-6 (IL-6)–inducible activation of STAT3 in human prostate cancer cells.

The SFN and its naturally occurring thio (iberverin, erucin,and berteroin), sulfinyl (iberin and alyssin), and sulfonyl (cheirolin, erysolin, and alyssin sulfone) analogues were purchased from LKT Laboratories. Stock solution of SFN and its analogues was prepared in DMSO and diluted with fresh medium immediately before use. An equal volume of DMSO (final concentration of <0.1%) was added to controls. Cell culture reagents, including medium, fetal bovine serum, and antibiotic mixture, were purchased from Life Technologies, whereas IL-6 was obtained from Sigma. Antibodies against phospho-STAT3 (pSTAT3; Tyr⁷⁰⁵), phospho–Janus-activated kinase 2 (pJAK2; Tyr^1007/1008), total STAT3, total JAK2, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase were obtained from Cell Signaling; antibodies against Mcl-1 and cyclin D1 were from Santa Cruz Biotechnology; anti–Bcl-2 antibody was from DakoCytomation; and anti-survivin antibody was from Novus Biologicals. The pSTAT3 (Tyr⁷⁰⁵) antibody used for immunofluorescence microscopy was from Santa Cruz Biotechnology. The STAT3-targeted small interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology. A control nonspecific siRNA (UUCUCCGAACGUGUCACGUdTdT) was from Qiagen. The sequence of the STAT3 siRNA is not revealed by the supplier. SYTOX Green and Alexa Fluor 568 goat anti-rabbit antibody were from Invitrogen.

Prostate cancer cell lines DU145, LNCaP, PC-3, and CWR22Rv1 were purchased from the American Type Culture Collection. Cell line authentication was done by analysis of known genetic differences (e.g., expression of androgen receptor and p53, and analysis of androgen responsiveness). The DU145, LNCaP, and PC-3 cells were cultured as described by us previously (20, 26). The CWR22Rv1 cells were cultured as recommended by the supplier.

Lysates from control and SFN-treated prostate cancer cells, and prostate tumor supernatants from control and SFN-treated transgenic adenocarcinoma of mouse prostate (TRAMP) mice were prepared as described by us previously (15, 27). Lysate/supernatant proteins were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. Immunoblotting was done as described by us previously (15, 27).

Total cellular RNA was extracted from control and SFN-treated LNCaP and DU145 cells using RNeasy kit (Invitrogen). Complementary DNA was synthesized from 1 to 2 μg of total RNA with reverse transcriptase and oligo(dT)₂₀. Reverse transcription-PCR was carried out using High Fidelity Taq polymerase (Invitrogen), gene-specific primers, and cDNA. The primers for JAK2 were 5′-GATGAGCAAGCTTTCTCACAAGC-3′ (forward) and 5-GCATGGCCCATGCCAACTGTTT-3′ (reverse). The primers for GAPDH were 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ (forward) and 5′-TCCTTGGAGGCCATGTGGGCCAT-3′ (reverse). Amplification conditions for JAK2 were 28 cycles at 94°C for 45 s, 58°C for 45 s, and 68°C for 45 s. Amplification conditions for GAPDH included 25 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s. The PCR products were resolved by 2% agarose gel prestained with ethidium bromide and visualized under UV illuminator.

Luciferase assay was done to determine the effect of SFN treatment on constitutive (DU145) and IL-6–induced (DU145 and LNCaP) transcriptional activity of STAT3. Briefly, cells (5 × 10⁴ per 12-well plates) were plated and allowed to attach by overnight incubation at 37°C. The cells were then cotransfected with 1 μg of pSTAT3-Luc plasmid encoding STAT3-responsive element and 0.1 μg of pRL-CMV plasmid using Fugene6 (Roche Applied Science) according to the manufacturer's recommendations. The pSTAT3-Luc plasmid was kindly provided by Dr. Bharat B. Aggarwal (University of Texas M.D. Anderson Cancer Center, Houston, TX; ref. 28). Twenty-four hours after transfection, the cells were treated with DMSO or SFN (20 or 40 μmol/L) for 12 h before stimulation with 10 ng/mL IL-6 for 12 h in the presence of SFN. Luciferase activity was determined using a luminometer and normalized against protein concentration.

Protein extracts from control and SFN-treated DU145 cells were prepared as described by Shin et al. (29). Proteins were resolved by 4% nondenaturing gel electrophoresis. Proteins were transferred onto polyvinylidene fluoride membrane and immunoblotted with the anti-STAT3 or anti-actin antibody as described above.

The effect of SFN treatment on nuclear localization of pSTAT3 was determined by immunocytochemistry. Briefly, DU145 or LNCaP cells (1 × 10⁵) were plated on coverslips, allowed to attach by overnight incubation, and exposed to DMSO (control) or SFN for 12 h. STAT3 activation was achieved by exposure to 10 ng/mL IL-6 for 15 min at the end of incubation period. The cells were fixed with 2% paraformaldehyde for 1 h at room temperature and permeabilized with 0.05% Triton X-100 for 5 min. The cells were then incubated with PBS supplemented with 0.5% bovine serum albumin and 0.15% glycine for 1 h followed by overnight incubation with anti-pSTAT3 antibody at 4°C. The cells were then treated with 2 μg/mL of Alexa Fluor 568–conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. After washing with PBS, the cells were counterstained with 0.5 μmol/L SYTOX Green for 3 min at room temperature. Subsequently, the cells were mounted and observed under a Leica DC300F fluorescence microscope at ×100 magnification.

The DU145 cells were seeded in six-well plates and transfected at 50% confluency with 100 nmol/L of control siRNA or STAT3-targeted siRNA using Oligofectamine according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were treated with DMSO (control) or specified concentrations of SFN for 24 h. The cells were then collected and processed for immunoblotting of total STAT3 and analysis of cytoplasmic histone-associated DNA fragmentation. Cytoplasmic histone-associated DNA fragmentation assay was done using a kit from Roche Applied Sciences according to the manufacturer's instructions.

The STAT3 activation is caused by phosphorylation at Tyr⁷⁰⁵ (23, 24). Initially, we did immunoblotting for pSTAT3 (Tyr⁷⁰⁵) using lysates from a panel of prostate cancer cell lines (CWR22Rv1, DU145, PC-3, and LNCaP) to determine constitutive activation of this transcription factor. Constitutive pSTAT3 was detectable only in the DU145 cell line with loading of up to 80 μg of the lysate protein (Fig. 1A). A 30-minute exposure of serum-starved (12-hour starvation) cells to 10 ng/mL IL-6, a known activator of STAT3 (30, 31), resulted in a robust increase in Tyr⁷⁰⁵ phosphorylation of STAT3 in DU145 and LNCaP cells (Fig. 1A, lanes 2 and 4). A modest increase in pSTAT3 level following IL-6 treatment was also observed in the CWR22Rv1 cell line but not in the PC-3 cells (Fig. 1A). Total STAT3 protein was detectable in every cell line except PC-3 (Fig. 1A). We selected DU145 and LNCaP cells to determine the effect of SFN treatment on constitutive and inducible activation of STAT3. As can be seen in Fig. 1B, SFN treatment reduced levels of pSTAT3 in DU145 cells in a concentration- and time-dependent manner. For example, the level of pSTAT3 was reduced by about 80% to 90% by a 24-hour exposure of DU145 cells to 20 and 40 μmol/L SFN (Fig. 1B). The SFN-mediated suppression of Tyr⁷⁰⁵ phosphorylation of STAT3 was not explained by a decrease in its protein level. Phosphorylation of STAT3 is mediated by different kinases, including JAK2 (23, 30, 31). We did immunoblotting for pJAK2 (Tyr^1007/1008) to determine if SFN-mediated inhibition of STAT3 activation was due to suppression of JAK2 phosphorylation. The level of pJAK2 was also reduced in a concentration- and time-dependent manner on exposure of DU145 cells to SFN, especially at the 12- and 24-hour time points (Fig. 1B). Unlike STAT3, however, suppression of JAK2 phosphorylation by SFN treatment correlated with a marked decrease in its protein level (Fig. 1B). We did reverse transcription-PCR to test whether SFN-mediated decline in JAK2 protein level was due to its reduced transcription. The level of JAK2 mRNA, after correction for GAPDH control, was decreased by approximately 70% to 90% in DU145 and LNCaP cells treated for 24 hours with 40 μmol/L SFN (results not shown). These results indicated that SFN treatment reduced transcription of JAK2.

Next, we tested the possibility whether SFN treatment affected IL-6–induced activation of STAT3. For these experiments, DU145 and LNCaP cells were treated with 40 μmol/L SFN for 24 hours and stimulated with 10 ng/mL IL-6 for the indicated time periods at the end of incubation period. The IL-6–induced phosphorylation of JAK2 as well as STAT3 was suppressed in both DU145 (Fig. 1C) and LNCaP cells (Fig. 1D). However, the LNCaP cell line (Fig. 1D) was markedly more sensitive to SFN-mediated inhibition of IL-6–inducible JAK2 and STAT3 phosphorylation as well as downregulation of total JAK2 protein expression compared with DU145 cells (Fig. 1C). The results were comparable if the cells were cotreated with SFN and IL-6 for 24 hours (data not shown). Collectively, these results indicated that SFN treatment inhibited constitutive as well as IL-6–induced phosphorylation of STAT3 by reducing the levels of total JAK2 and pJAK2.

We did luciferase reporter assay to determine the effect of SFN treatment on transcriptional activity of STAT3. In DU145 cells, a 24-hour treatment with 40 μmol/L SFN resulted in a statistically significant decrease in STAT3-dependent luciferase activity (∼58% inhibition compared with DMSO-treated control; Fig. 2A). As expected, transcriptional activity of STAT3 was modestly increased in the presence of IL-6 (10 ng/mL, 12-hour exposure in the presence of SFN). The IL-6–mediated increase in STAT3-associated luciferase activity was completely abolished in the presence of SFN (Fig. 2A). Consistent with immunoblotting data (Fig. 1D), the IL-6–stimulated STAT3-dependent luciferase activity in LNCaP cells was completely abolished by a 24-hour exposure to 20 and 40 μmol/L SFN (Fig. 2B). These results indicated that SFN treatment inhibited constitutive and IL-6–induced transcriptional activity of STAT3 in DU145 and LNCaP cells.

Phosphorylation at Tyr⁷⁰⁵ results in homodimerization of STAT3 or heterodimerization with other STATs, which enables nuclear translocation of STAT3 for binding to specific sequences of target genes (23, 28, 31). We designed experiments using DU145 cells to determine whether SFN treatment affected homodimerization and nuclear translocation of STAT3. As shown in Fig. 2C, electrophoresis followed by immunoblotting revealed time-dependent reduction in level of STAT3 homodimer in DU145 cells on treatment with 40 μmol/L SFN.

Figure 3A depicts immunostaining for constitutive pSTAT3 (red fluorescence) and SYTOX Green–associated nuclear staining (green fluorescence) in DU145 cells following 12-hour treatment with DMSO (control) or the indicated concentrations of SFN. In DU145 cells, pSTAT3 was predominantly localized in the nucleus as evidenced by yellow-orange staining due to merging of red and green fluorescence. Nuclear level of pSTAT3 was decreased markedly in cells treated with 20 and 40 μmol/L SFN (Fig. 3A). As expected, a 15-minute exposure of DU145 cells to 10 ng/mL IL-6 resulted in increased nuclear level of pSTAT3 as evidenced by more intense yellow-orange staining (Fig. 3B) compared with unstimulated DU145 cells (Fig. 3A). The IL-6–stimulated nuclear localization of pSTAT3 was also inhibited in the presence of SFN in both DU145 (Fig. 3B) and LNCaP (Fig. 3C) cells at each concentration. Nuclear staining for pSTAT3 in unstimulated LNCaP cells was practically undetectable (results not shown), which is consistent with the immunoblotting data (Fig. 1A). These results showed inhibition of constitutive and IL-6–induced dimerization and/or nuclear localization of pSTAT3 in the presence of SFN in DU145 and LNCaP cells.

STAT3 is known to regulate expression of various genes involved in cell proliferation and apoptosis, including Mcl-1, Bcl-2, cyclin D1, and survivin (23, 28, 31). To test whether SFN-mediated suppression of STAT3 activation resulted in downregulation of its target gene products, we did immunoblotting for above-mentioned proteins using lysates from DU145 and LNCaP cells treated with DMSO (control) and SFN. The levels of Mcl-1, Bcl-2, cyclin D1, and survivin proteins were altered on treatment with SFN in both cell lines, albeit with different kinetics or cell line specificity. The SFN-mediated downregulation of Bcl-2 and cyclin D1 protein levels was clearly evident in both cell lines, especially at the 12- to 24-hour time points (Fig. 4A and B). The SFN treatment decreased protein level of survivin at 6- to 12-hour time points in DU145 cells (Fig. 4A) and at 24-hour time point in the LNCaP cell line (Fig. 4B). We did additional immunoblotting analyses to test whether expression of the above proteins was regulated by IL-6/STAT3 in DU145 and LNCaP cells. As can be seen in Fig. 4C, expression of Mcl-1, Bcl-2, and cyclin D1 proteins was increased by stimulation with IL-6. The effect on survivin protein level was inconsistent (results not shown). The IL-6–stimulated induction of Mcl-1, Bcl-2, and cyclin D1 protein expression was attenuated to varying extent in the presence of SFN (Fig. 4C). These results indicated that the SFN-mediated suppression of STAT3 activation was accompanied by downregulation of some of its target gene products.

To test biological significance of SFN-mediated suppression of STAT3 activation in our model, we determined apoptosis induction by SFN in LNCaP cells treated for 24 hours with 20 and 40 μmol/L in the presence or absence of 10 ng/mL IL-6 (24-hour treatment). As shown in Fig. 5A, the SFN treatment resulted in a concentration-dependent and statistically significant increase in cytoplasmic histone-associated DNA fragmentation, which is a highly sensitive and reliable technique for quantitation of apoptotic cell death. The apoptotic DNA fragmentation resulting from SFN treatment was partially but significantly abrogated in IL-6–stimulated cells (Fig. 5A). Consistent with these results, SFN-mediated cleavage of poly(ADP-ribose) polymerase and caspase-3 was markedly inhibited in the presence of IL-6 in LNCaP cells (Fig. 5B).

We determined the effect of STAT3 protein knockdown on SFN-induced apoptosis using DU145 cells. The level of STAT3 protein was reduced by ∼70% on transient transfection of DU145 cells with STAT3-targeted siRNA in comparison with cells transfected with a control nonspecific siRNA (Fig. 5C, inset). Knockdown of STAT3 protein in DU145 cells resulted in a modest yet statistically significant increase in SFN-induced cytoplasmic histone-associated DNA fragmentation (Fig. 5C). Collectively, these results indicated that STAT3 activation conferred partial protection against SFN-induced apoptosis in prostate cancer cells. It is also clear that the DU145 cell line is markedly more resistant to SFN-induced apoptotic DNA fragmentation (Fig. 5C) compared with LNCaP cells (Fig. 5A). Because STAT3 protein knockdown resulted in only modest increase in SFN-induced apoptosis in DU145 cells, our interpretation is that differences in constitutively active STAT3 levels between these cell lines may only partially account for differential sensitivity of these cells to SFN-induced apoptosis. At the same time, the possibility that complete knockdown of STAT3 protein results in a greater sensitization of DU145 cells to SFN-induced apoptosis cannot be fully discarded.

We have shown previously that oral administration of SFN retards development of prostate cancer in TRAMP mice (15). We used the prostate tumor tissues harvested from representative control and SFN-treated TRAMP mice to determine effect on total STAT3/JAK2 and pSTAT3/pJAK2 levels. As shown in Fig. 5D, the prostate tumors from SFN-treated TRAMP mice exhibited a decrease in levels of pSTAT3 (∼20% decrease), total STAT3 (∼30% decrease), and total JAK2 (∼70% decrease) compared with controls. Expression of pJAK2 was not detectable in the prostate tumors of either control or SFN-treated TRAMP mice (results not shown). These results indicated that SFN administration reduced levels of total STAT3/JAK2 and pSTAT3 in prostate tumors of TRAMP mice.

We used naturally occurring thio, sulfinyl, and sulfonyl analogues of SFN (refer to Table 1 for chemical formula of the analogues) to determine possible effect of oxidation state of sulfur and alkyl chain length on thioalkyl isothiocyanate–mediated inhibition of STAT3/JAK2 phosphorylation. As can be seen in Fig. 6, inhibition of JAK2 and STAT3 phosphorylation as well as JAK2 downregulation was also observed in cells treated for 24 hours with SFN analogues. Although a clear structure-activity relationship was not discernible, suppression of STAT3 phosphorylation at 20 μmol/L concentration was more pronounced for SFN compared with other compounds. In general, the sulfonyl derivatives (cheirolin, erysolin, and alyssin sulfone) were relatively weak suppressors of STAT3 phosphorylation compared with SFN despite potent inhibition of JAK2 phosphorylation. These results suggest that the sulfonyl derivatives might activate other kinases (e.g., Src) to counteract inhibition of JAK2 phosphorylation.

STAT3, a nuclear transcription factor belonging to the seven-member STAT gene family of transcription factors, plays an important role in promotion of growth and progression of prostate cancer (24, 25, 32–35). STAT3 is constitutively active in clinical human prostate cancer (33, 35). For example, Dhir et al. (33) observed significantly higher levels of constitutively active STAT3 in both prostate carcinomas and the matched normal prostate tissues adjacent to tumors compared with the normal prostates from donors without prostate cancer. However, no correlation was noted between STAT3 activation and Gleason grade or serum prostate-specific antigen levels (33). Several reports also indicate that STAT3 promotes proliferation and inhibits apoptosis in cultured prostate cancer cells (24, 36). Moreover, STAT3 is implicated in transition of hormone-sensitive prostate cancer to androgen-independent state (also referred to as “castration resistant” or “hormone refractory”; refs. 25, 36), which is highly aggressive and resistant to chemotherapy (37). Accordingly, agents that are relatively safe but could suppress activation of STAT3 are highly attractive for both prevention of prostate cancer and treatment of castration-resistant disease. The present study shows that SFN, a promising cancer chemopreventive agent, inhibits both constitutive and IL-6–induced activation of STAT3 in human prostate cancer cells regardless of their androgen responsiveness. The SFN-mediated suppression of STAT3 activation in human prostate cancer cells is evident at pharmacologically relevant concentrations based on our own pharmacokinetic data. We have shown previously that gavage with a single dose of 6 μmol SFN results in peak plasma concentration of ∼19 μmol/L in mice (15).

Activation of STAT3 entails phosphorylation on a critical tyrosine residue (Tyr⁷⁰⁵), which is mediated by different kinases, including JAKs, Rac1, and Src (23, 28, 31, 38). The IL-6–mediated activation of STAT3 is regulated by JAKs through cytoplasmic domain of gp130 of the IL-6 receptor (39). The present study reveals that SFN-mediated inhibition of STAT3 activation in prostate cancer cells is accompanied by suppression of constitutive as well as IL-6–inducible Tyr^1007/1008 phosphorylation of JAK2. The time course kinetics of SFN-mediated suppression of pJAK2 mirrors that of pSTAT3 inhibition. The mechanism by which SFN inhibits JAK2 phosphorylation seems to involve transcriptional repression of JAK2, leading to a reduction in its protein level. Inhibition of STAT3 activation in DU145 cells by a naturally occurring structural analogue of SFN (phenethyl isothiocyanate) was reported recently during completion of our work (40). However, unlike our results, the phenethyl isothiocyanate–mediated suppression of STAT3 activation was not due to a decrease in level of JAK2 protein (40). These observations underscore mechanistic differences between structurally related SFN and phenethyl isothiocyanate. Recent studies from our laboratory have documented additional unique mechanistic differences between SFN and phenethyl isothiocyanate. For example, SFN causes autophagy that serves to protect prostate cancer cells against apoptosis (41). On the other hand, autophagy induction seems to contribute to the overall cell death caused by phenethyl isothiocyanate (42).

The Tyr⁷⁰⁵ phosphorylation of STAT3 causes its homodimerization (28, 31). The STAT3 dimer translocates to the nucleus for binding to specific DNA sequences in the promoter of target genes (28, 31). The present study indicates that the SFN-mediated inhibition of STAT3 phosphorylation suppresses its transcriptional activity as evidenced by (a) inhibition of constitutive and IL-6–inducible STAT3-dependent luciferase reporter activity in the presence of SFN, (b) a reduction in level of constitutive STAT3 homodimer in SFN-treated DU145 cells, (c) a decrease in constitutive and IL-6–stimulated nuclear translocation of pSTAT3 on treatment with SFN, and (d) suppression of many STAT3-regulated gene products (e.g., cyclin D1 and survivin) in SFN-treated DU145 and LNCaP cells.

The present study also addresses the question of whether suppression of STAT3 activation contributes to apoptotic cell death resulting from SFN exposure. The IL-6–stimulated activation of STAT3 confers partial yet significant protection against SFN-induced apoptosis in both LNCaP (Fig. 5A) and DU145 cells (results not shown). Knockdown of STAT3 results in only modest increase in SFN-induced apoptosis in DU145 cells, which could be attributed to incomplete knockdown of the STAT3 protein. Alternatively, the possibility that suppression of STAT3 only partially accounts for SFN-induced apoptosis in prostate cancer cells is also likely. To this end, we have shown previously that SFN treatment causes generation of reactive oxygen species, leading to activation of mitochondria-mediated apoptosis in PC-3 and LNCaP human prostate cancer cells (20, 21).

In conclusion, the present study provides experimental evidence to indicate that cancer chemopreventive agent SFN causes suppression of constitutive as well as IL-6–induced activation of STAT3 in human prostate cancer cells irrespective of their androgen responsiveness. Moreover, the IL-6–mediated activation of STAT3 activation confers partial yet statistically significant protection against SFN-induced apoptosis.

No potential conflicts of interest were disclosed.

We thank Dr. Bharat B. Aggarwal for the generous gift of pSTAT3-Luc plasmid.

Grant Support: USPHS grants CA115498-05 and CA101753-07 awarded by the National Cancer Institute.

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.