SFN Administration Research Articles

EGFR/MAPK signaling pathway acts as a potential therapeutic target for sulforaphane-rescued heart tube malformation induced by various concentrations of PhIP exposure

Background2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyrimidine (PhIP) is a known carcinogen generated mainly from cooking meat and environmental pollutants. It is worth exploring the potential of natural small-molecule drugs to protect against adverse effects on embryonic development. PurposeIn this study, we investigated the potential toxicological effects of PhIP on embryonic heart tube formation and the effect of Sulforaphane (SFN) administration on the anti-toxicological effects of PhIP on embryonic cardiogenesis. Study design and methodsFirst, the chicken embryo model was used to investigate the different phenotypes of embryonic heart tubes induced by various concentrations of PhIP exposure. We also proved that SFN rescues PhIP-induced embryonic heart tube malformation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR) and flow cytometry experiments were employed to explore the mechanisms by which SFN protects cardiac cells from oxidative damage in the presence of PhIP. We used RNA-seq analysis, molecular docking, in situ hybridization, cellular thermal shift assay and solution nuclear magnetic resonance spectroscopy to explore whether SFN protects cardiogenesis through the EGFR/MAPK signaling pathway. ResultsThe study showed that PhIP might dose-dependently interfere with the C-looping heart tube (mild) or the fusion of a pair of bilateral endocardial tubes (severe) in chick embryos, while SFN administration prevented cardiac cells from oxidative damage in the presence of high-level PhIP. Furthermore, we found that excessive reactive oxygen species (ROS) production and subsequent apoptosis were not the principal mechanisms by which low-level PhIP induced malformation of heart tubes. This is due to PhIP-disturbed Mitogen-activated protein kinase (MAPK) signaling pathway could be corrected by SFN administration. ConclusionsThis study provided novel insight that PhIP exposure could increase the risk of abnormalities in early cardiogenesis and that SFN could partially rescue various concentrations of PhIP-induced abnormal heart tube formation by targeting EGFR and mediating EGFR/MAPK signaling pathways.

Sulforaphane Attenuates Isoproterenol-Induced Myocardial Injury in Mice.

The development of isoproterenol- (ISO-) induced oxidative stress in the myocardium results in myocardial necrosis. Sulforaphane (SFN-0.4% of sulforaphane from standardized broccoli sprout extract) possesses chemoprotective, antidiabetic, and antibacterial activities and is also active against cardiovascular-related problems due to its antioxidant properties. This study was designed to investigate the cardioprotective effect of SFN against isoproterenol-induced myocardial injury in mice. Healthy male Swiss albino mice weighing 20–30 g were used in this study. These mice were randomly divided into five groups (n = 6). All the mice in the experimental groups received isoproterenol (5 mg/kg bw, via i.p.) consecutively for 2 days. The mice were treated with SFN (4 mg/kg bw) and α-tocopherol (TCF) (10 mg/kg bw) by oral gavage for 1-7 days as pre- and posttreatment for the prophylactic and treatment groups, respectively. On day 10, the following parameters were studied: heart weight to body weight ratio, antioxidant parameters, and cardiac markers; and mitochondrial enzymes were estimated for cardioprotection. Administration of isoproterenol in mice showed an increased level of serum cardiac markers and heart mitochondrial ATPase enzymes. An increased level of myocardial thiobarbituric acid-reactive substance and decreased levels of endogenous antioxidant enzymes indicated that oxidative stress is induced by isoproterenol in the myocardium. The administration of SFN in mice restored the levels of all biochemical parameters to near-normal levels. Histopathological studies further confirmed the protective effect of sulforaphane. This study concluded that treatment with SFN boosts the endogenous antioxidant activity and prevents isoproterenol-induced myocardial injury.

A Novel Sulforaphane-Regulated Gene Network in Suppression of Breast Cancer-Induced Osteolytic Bone Resorption.

Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer-induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. In vitro osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of runt-related transcription factor 2 (RUNX2) and nuclear factor-κB and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of NFKB1 Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating in vivo suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer-induced osteolytic bone resorption in vitro and in vivo.

Abstract 5070: Sulforaphane is a novel inhibitor of breast cancer-induced osteolytic bone resorption

Abstract Bone is the preferred site for metastatic spread in women with advanced breast cancer, and skeletal complications is associated with high morbidity and mortality. Nearly 70% of luminal-type breast cancer patients with metastases to bone experience skeletal complications due to enhanced osteoclastogenesis (osteoclast activation) that increases bone resorption, pain, pathological fractures, spinal cord compression, and hypercalcemia. The present study was designed to determine the effect of a cruciferous vegetable component (Sulforaphane; SFN) on breast cancer-induced osteoclastogenesis. Osteoclast differentiation in mouse bone marrow monocytes (BMM) was inhibited significantly upon the addition of 10% conditioned-media (CM) from SFN-treated breast cancer cells belonging to different subtypes, including MDA-MB-231, MCF-7, and SK-BR-3, in comparison with corresponding control. PCR-based gene expression profiling identified a common set of genes downregulated by SFN treatment compared to vehicle-treated control in MDA-MB-231, MCF-7, and SK-BR-3 cells, including osteoclastogenesis promoting transcription factors (RUNX2, NF-κB, and SOX-9) and certain soluble molecules (MMP9, TNFα, and Cathepsin K). Many of these gene expression changes were confirmed in MDA-MB-231, MCF-7, and SK-BR-3 cells by RT-PCR or western blotting. To determine the in vivo efficacy, SFN was administrated orally (1 mg per mouse; three times/week) to athymic mice intracardially injected with MDA-MB-231-Luc cells. SFN administration significantly inhibited the multiplicity of bone metastases and increased the bone volume relative to total volume. SFN-mediated prevention of osteoclastogenesis was associated with a significant decrease in TRAP-positive osteoclasts in bones and the levels of Cathepsin K, IL-8, and RANKL in serum. Altogether, this study demonstrates, for the first time to the best of our knowledge, that SFN is a potent inhibitor of breast cancer-induced osteoclastogenesis and bone resorption in vitro and in vivo. This study was partly funded by a pilot project grant from the UPMC Hillman Cancer (NCI grant P30 CA047904; Robert L. Ferris- Principal Investigator) and RO1 CA142604 and CA129347 awarded by the National Cancer Institute (Shivendra Singh- Principal Investigator). Citation Format: Subrata K. Pore, Joseph D. Latoche, Carolyn J. Anderson, Juraj Adamik, Deborah L. Galson, Kurt R. Weiss, Boeun Lee, Rebecca J. Watters, Prashant N. Kumta, Shivendra V. Singh. Sulforaphane is a novel inhibitor of breast cancer-induced osteolytic bone resorption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5070.

The effect of sulforaphane on oxidative stress and inflammation in rats with toxic hepatitis induced by acetaminophene.

The aim of the present study was to reveal the possible effect of sulforaphane on oxidative stress and inflammation in rats liver with toxic hepatitis induced by acetaminophene. Sulforaphane is a compound with high antioxidant properties. Acetaminophen, which is a para-aminophenol derivative, can lead to fatal hepatic necrosis with direct hepatotoxic effects at high doses. Thirty six male Sprague-Dawley rats were randomly divided into four groups. Control group (n = 9) was fed with standard rat chow and water for 3 days. Group APAP (n = 9) received a single dose acetaminophen 1 g/kg by oral gavage in addition to standard chow and water. Group SFN (n = 9) received sulforaphane 500 μg/kg by oral gavage in addition to standard chow and water for 3 days. Group APAP+SFN (n = 9) received sulforaphane 500 μg/kg and a single dose acetaminophen 1 g/kg by oral gavage in addition to standard chow and water. Acetaminophen was administered three hours after SFN administration. Neopterin, MDA, AST, ALT and CRP levels of group APAP were significantly increased compared to control group. GSH level of group APAP was significantly lower than in the control group. Sulforaphane is a protective agent against acetaminophen-induced liver damage and it can be added in the treatment protocol (Tab. 1, Fig. 5, Ref. 51).

Sulforaphane activates the cerebral vascular Nrf2–ARE pathway and suppresses inflammation to attenuate cerebral vasospasm in rat with subarachnoid hemorrhage

Nrf2–ARE pathway reportedly plays a protective role in several central nervous system diseases. No study has explored the role of the Nrf2–ARE pathway in cerebral vasospasm(CVS) after subarachnoid hemorrhage(SAH). The purpose of the present study was to investigate the activation of the cerebral vascular Nrf2–ARE pathway and to determine the potential role of this pathway in the development of CVS following SAH. We investigated whether the administration of sulforaphane (SFN, a specific Nrf2 activator) modulated vascular caliber, Nrf2-ARE pathway activity, proinflammatory cytokine expression, and clinical behavior in a rat model of SAH. A two-hemorrhage protocol was used to generate an animal model of SAH in male Sprague-Dawley rats. Administration of SFN to these rats following SAH enhanced the activity of the Nrf2-ARE pathway and suppressed the release of proinflammatory cytokines. Vasospasm was markedly attenuated in the basilar arteries after SFN therapy. Additionally, SFN administration significantly ameliorated two behavioral functions disrupted by SAH. These results suggest that SFN has a therapeutic benefit in post-SAH, and this may be due to elevated Nrf2-ARE pathway activity and inhibition of cerebral vascular proinflammatory cytokine expression.

Abstract 822: Role of c-Myc in prostate cancer stem-like cell inhibition by sulforaphane

Abstract Chemopreventive efficacy of D,L-sulforaphane (SFN) against prostate cancer has been shown in a transgenic mouse model but the underlying mechanism is not fully understood. The present study demonstrates that c-Myc, which is an oncogenic transcription factor frequently upregulated in human prostate cancer, is a molecular target of SFN. Exposure of human prostate cancer cells (LNCaP, C4-2, and PC-3) and a cell line derived from prostate adenocarcinoma of a transgenic mouse (Myc-CaP) to SFN resulted in a marked decrease in c-Myc protein level. Naturally-occurring thio, sulfinyl, and sulfonyl analogues of SFN were also effective in causing suppression of c-Myc protein level. The c-Myc is a known regulator of cellular metabolism but basal glycolysis was not altered by SFN treatment in empty vector transfected or in c-Myc overexpressing PC-3 cells. The effect of SFN treatment on prostate cancer stem-like cells (pCSC) was also determined as c-Myc is implicated in their maintenance. Analysis of aldehyde dehydrogenase 1 activity, CD49f+ fraction, and sphere forming efficiency revealed dose-dependent inhibition of pCSC in SFN-treated cells. The pCSC inhibition by SFN was partially but significantly attenuated by overexpression of c-Myc. Expression profiling revealed upregulation of a set of stemness genes by c-Myc overexpression but their downregulation by SFN treatment. Finally, oral administration of SFN to Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice resulted in a marked decrease in c-Myc protein expression in the adenocarcinoma. In conclusion, this study indicates that c-Myc downregulation contributes to pCSC inhibition by SFN. This investigation was supported by grant CA115498 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Avani R. Vyas, Shivendra V. Singh. Role of c-Myc in prostate cancer stem-like cell inhibition by sulforaphane. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 822.

Nrf2 sensitizes prostate cancer cells to radiation via decreasing basal ROS levels.

Androgen deprivation therapy (ADT) was reported to lower basal ROS level in prostate cancer (PCa) and to sensitize PCa to radiation. We aimed to seek for the underlying molecular mechanism and to develop novel additive treatments to ADT in this regard. We simulated human androgen milieu in vitro and tested the ROS level in PCa cells undergoing ADT. We also tested the Nrf2 level in PCa cells with or without ADT. Genetic and pharmaceutical upregulation of Nrf2 was applied in vitro and in vivo in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with or without castration to investigate whether Nrf2 overexpression supplemented the effect of ADT in PCa. We first discovered that androgen deprivation increased basal ROS level in PCa cells with AR expression. We then found that genetic Nrf2 upregulation lowered basal ROS similar to ADT. Also, SFN sensitized PCa cell to radiation via upregulation of Nrf2. We then found that Nrf2 level in control TRAMP groups was lower than castration or SFN groups. The SFN treated TRAMP mice showed similar level of Nrf2 to castration. Genetic and pharmaceutical upregulation of Nrf2 lowered the ROS in PCa cells and sensitized PCa cells to radiation similar to ADT, implicating possible administration of SFN in place of ADT for PCa patients requiring radiotherapy.

Abstract 3695: Augmentation of D,L-sulforaphane-mediated prostate cancer chemoprevention by pharmacologic inhibition of autophagy using chloroquine in a transgenic mouse model.

Abstract D,L-Sulforaphane (SFN) is a synthetic racemic analogue of broccoli constituent L-sulforaphane with in vivo evidence of cancer chemopreventive effect in rodent models. For example, we showed previously that oral administration of 6 μmol (about 1 mg/mouse) SFN (3x/week) modestly inhibited incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated cancer, but not poorly-differentiated (advanced) prostate cancer, in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice in association with apoptosis induction. Previous studies from our laboratory have also revealed that SFN treatment causes autophagy in cultured human prostate cancer cells, which serves to inhibit apoptotic cell death by delaying release of cytochrome c due to sequestration of mitochondria in autophagosomes. However, the in vivo significance of these cellular observations was unclear. The present study demonstrates, for the first time, in vivo augmentation of SFN-mediated prostate cancer chemoprevention by pharmacologic suppression of autophagy using chloroquine (CQ). A total of 128 four-week old TRAMP mice were randomized into one of the following four groups: (1) control (n=32), (2) SFN (n=32), (3) CQ (n=35), and (4) SFN + CQ (n=29). Termination of the experiment was planned after 18 weeks of treatment, but early sacrifice was necessary for some mice in each group (mostly from groups 1-3) due to a variety of reasons but not unique to any treatment group, including weight loss, premature death, morbidity, or early tumor onset. Nevertheless, % mice with prostate weight of >1g, an indicator of advanced (poorly-differentiated) prostate cancer, was significantly lower in the SFN + CQ group compared with control (P=0.006 by Fisher's exact test), SFN alone (P=0.017), and CQ alone group (P=0.072). Likewise, the incidence and burden of microscopic poorly-differentiated prostate cancer was significantly lower in the mice of the SFN + CQ group compared with control. Transmission electron microscopy confirmed in vivo autophagy induction by SFN administration in the dorsolateral prostate. Augmentation of SFN-mediated prostate cancer chemoprevention by CQ was associated with reduced cell proliferation. Plasma proteomics indicated protein level changes unique to the SFN + CQ combination compared with other groups (control or SFN), including hemopexin, serpina1c protein, and fructose-bisphosphate aldolase A isoform precursor. Cluster analysis of proteomics data revealed significant enrichment for gene ontology (GO) terms proteasome and threonine protease (2.8), protease inhibitor (2.7), and protein-lipid complex (2.6). These results merit determination of the efficacy of SFN + CQ combination for prevention of prostate cancer in a clinical setting. This investigation was supported by the NCI grant CA115498-07. Citation Format: Avani R. Vyas, Eun-Ryeong Hahm, Julie A. Arlotti, Dhimant Desai, Shantu Amin, Shivendra V. Singh. Augmentation of D,L-sulforaphane-mediated prostate cancer chemoprevention by pharmacologic inhibition of autophagy using chloroquine in a transgenic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2013-3695

Amelioration of Alzheimer’s disease by neuroprotective effect of sulforaphane in animal model

Pathophysiological evidences of AD have indicated that aggregation of Aβ is one of the principal causes of neuronal dysfunction, largely by way of inducing oxidative stresses such as free radical formation. We hypothesized that the known antioxidative attribute of SFN could be harnessed in Alzheimer’s treatment. SFN is an indirect, potent antioxidant derived from broccoli that has previously been found to stimulate the Nrf2-ARE pathway and facilitate several other cytoprotective mechanisms. In this study, administration of SFN ameliorated cognitive function of Aβ-induced AD acute mouse models in Y-maze and passive avoidance behavior tests. Interestingly, we found that the therapeutic effect of SFN did not involve inhibition of Aβ aggregation. While the exact mechanism of interaction of SFN in AD has not yet been ascertained, our results suggest that SFN can aid in cognitive impairment and may protect the brain from amyloidogenic damages.Abbreviations: Aβ, amyloid-β; AD, Alzheimer’s disease; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; SFN, sulforaphane; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum; ICR, imprinting control region; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DW, deionized water; ThT, thioflavin T; PA, passive avoidance; ARE, antioxidant response element.

Notch Activation Is Dispensable for D, L-Sulforaphane-Mediated Inhibition of Human Prostate Cancer Cell Migration

D, L-Sulforaphane (SFN), a synthetic racemic analog of broccoli constituent L-sulforaphane, is a highly promising cancer chemopreventive agent with in vivo efficacy against chemically-induced as well as oncogene-driven cancer in preclinical rodent models. Cancer chemopreventive effect of SFN is characterized by G2/M phase cell cycle arrest, apoptosis induction, and inhibition of cell migration and invasion. Moreover, SFN inhibits multiple oncogenic signaling pathways often hyperactive in human cancers, including nuclear factor-κB, Akt, signal transducer and activator of transcription 3, and androgen receptor. The present study was designed to determine the role of Notch signaling, which is constitutively active in many human cancers, in anticancer effects of SFN using prostate cancer cells as a model. Exposure of human prostate cancer cells (PC-3, LNCaP, and/or LNCaP-C4-2B) to SFN as well as its naturally-occurring thio-, sulfinyl-, and sulfonyl-analogs resulted in cleavage (activation) of Notch1, Notch2, and Notch4, which was accompanied by a decrease in levels of full-length Notch forms especially at the 16- and 24-hour time points. The SFN-mediated cleavage of Notch isoforms was associated with its transcriptional activation as evidenced by RBP-Jk-, HES-1A/B- and HEY-1 luciferase reporter assays. Migration of PC-3 and LNCaP cells was decreased significantly by RNA interference of Notch1 and Notch2, but not Notch4. Furthermore, SFN-mediated inhibition of PC-3 and LNCaP cell migration was only marginally affected by knockdown of Notch1 and Notch2. Strikingly, SFN administration to Transgenic Adenocarcinoma of Mouse Prostate transgenic mice failed to increase levels of cleaved Notch1, cleaved Notch2, and HES-1 proteins in vivo in prostatic intraepithelial neoplasia, well-differentiated carcinoma or poorly-differentiated prostate cancer lesions. These results indicate that Notch activation is largely dispensable for SFN-mediated inhibition of cell migration, which should be viewed as a therapeutic advantage as Notch activation is frequent in human prostate cancers.

Abstract 866: D,L-Sulforaphane-induced apoptosis in human breast cancer cells is regulated by growth factor adaptor protein p66Shc

Abstract D,L-Sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, inhibits chemically-induced cancer in experimental rodents. SFN administration also suppresses cancer development in transgenic mouse models. Anticancer effect of SFN is associated with apoptosis induction but the mechanism of cell death is not fully understood. Using MDA-MB-231 (an estrogen-independent cell line) and MCF-7 (an estrogen-responsive cell line) human breast cancer cells as a model, we now demonstrate that the SFN-induced apoptosis is regulated by growth factor adapter protein p66Shc. Exposure of MDA-MB-231 and MCF-7 cells to SFN resulted in increased Ser36 phosphorylation of p66Shc. Immortalized mouse embryonic fibroblasts derived from p66Shc knockout mouse were significantly more resistant to SFN-mediated growth inhibition (judged by trypan blue dye exclusion assay) and apoptosis induction (judged by cytoplasmic histone-associated apoptotic DNA fragmentation) compared with embryonic fibroblasts from wild-type mouse. Moreover, transient transfection of MDA-MB-231 and MCF-7 cells with p66Shc-targeted siRNA conferred significant protection against SFN-induced cytoplasmic histone-associated DNA fragmentation. Protein level of prolyl isomerase 1 (Pin1), a protein implicated in mitochondrial translocation of p66Shc, was decreased on a 24-hour treatment with SFN in both MDA-MB-231 and MCF-7 cells. However, apoptosis induction resulting from SFN exposure was not affected by ectopic expression of Pin1 (MCF-7). In conclusion, the present study provides novel insights into the molecular circuitry of SFN-induced apoptosis involving p66Shc. This investigation was supported by the USPHS grant RO1 CA115498-05, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 866. doi:10.1158/1538-7445.AM2011-866

Sulphoraphane enhances aquaporin-4 expression and decreases spinal cord oedema following spinal cord injury

Background: Aquaporin-4 (AQP4) is a water channel protein and it is an important determinant of outcome after brain injury. Sulphoraphane (SFN) increases AQP4 levels with reduction of brain oedema at 3 days post-traumatic brain injury. However, little is known about the effect of SFN on AQP4 expression and oedema after spinal cord injury (SCI).Methods and procedures: The present study used a murine SCI model induced by compression injury. AQP4 protein level and mRNA level were detected by Western blot and by RT-PCR at 48 hours after SCI, respectively. In addition, immunohistochemical study was used to show AQP4 expression in the spinal cord segments and water content of the spinal cord segments were measured by wet : dry weight ratio.Main outcomes and results: This study shows that AQP4 level was decreased in the injured spinal cord segments at 48 hours following SCI. Post-injury administration of SFN increased AQP4 levels, which was accompanied by a significant reduction in spinal cord segment oedema at 48 hours post-injury.Conclusion: These findings suggest that the reduction of spinal cord oedema in response to SFN administration could be due, in part, to water clearance by AQP4 from the injured spinal cord segments.

Abstract 3787: D, L-Sulforaphane inhibits constitutive and interleukin 6-induced activation of signal transducer and activator of transcription 3 in human prostate cancer cells

Abstract D, L-Sulforaphane (SFN), a synthetic analogue of broccoli-derived L-isomer, inhibits viability of human prostate cancer cells in culture and in vivo. We also showed recently that oral administration of SFN prevents development of prostate cancer and pulmonary metastasis in a transgenic mouse model (TRAMP mice) by reducing cell proliferation and augmenting activity of natural killer cells. However, the mechanism underlying anticancer effect of SFN is not fully understood. We now demonstrate that SFN inhibits constitutive and interleukin 6 (IL6)-induced activation of signal transducer and activator of transcription 3 (STAT3), which is implicated in development and progression of prostate cancer. Growth suppressive concentrations of SFN (20 and 40 μmol/L) inhibited constitutive (DU145 cells) and IL6-induced (LNCaP cells) phosphorylation of STAT3 (Tyr705) as well as its upstream regulator Janus-activated kinase 2 (JAK2; Tyr1007/1008). Treatment of DU145 and LNCaP cells with SFN resulted in suppression of IL6-induced transcriptional activity of STAT3 as revealed by luciferase reporter assay and nuclear translocation of STAT3 as judged by immunofluorescence microscopy. The SFN-mediated inhibition of STAT3 activation was due to down-regulation of JAK2 mRNA expression in both cell lines. Levels of many STAT3-regulated gene products including Bcl-2, cyclin D1, and survivin were also down-regulated in SFN-treated cells. The IL6-mediated activation of STAT3 conferred partial but marked protection against SFN-induced apoptosis in LNCaP cells as evidenced by cleavage of poly-(ADP-ribose)-polymerase and caspase-3 and cytoplasmic histone-associated DNA fragmentation. In addition, siRNA knockdown of STAT3 in DU145 cells resulted in a modest yet statistically significant increase in SFN-induced apoptotic DNA fragmentation. The results of the present study demonstrate SFN-mediated inhibition of STAT3 activation in human prostate cancer cells. This investigation was supported by the National Cancer Institute grant CA115498-05. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3787.

NADPH quinone oxidoreductase induction by sulforaphane inhibits synovial hyperplasia and rheumatoid T cell proliferation. (50.15)

Abstract Sulforaphane (SFN), derived from cruciferous vegetables, has been previously shown to induce phase II enzyme and to have anti-carcinogenic activity. In this study, we investigated whether SFN regulates synoviocyte hyperplasia and T cell proliferation in rheumatoid arthritis (RA). SFN induced synoviocyte apoptosis by regulating Bcl-2/Bax expression and pAkt activity. In addition, SFN dose-dependently inhibited T cell proliferation and the production of IL-17 and TNF-alpha by RA mononuclear cells stimulated with anti-CD3 antibody, but enhanced the enzyme activities of NADPH quinone oxidoreductase-1 (NQO1), a representative phase II enzyme. SFN inhibition of synoviocyte survival was abrogated by co-treatment with curcumin, an inhibitor of NQO1 or by downregulation of NQO1 transcripts. The intra-peritoneal administration of SFN to mice with type II collagen (CII)-induced arthritis suppressed arthritis severity, anti-CII antibody levels, and T cell proliferative response to CII. The productions of IL-17, TNF-alpha, IL-6, and IFN-gamma from lymph node and spleen cells of these mice also were markedly reduced by SFN treatment. Moreover, in mice with anti-CII antibody-induced arthritis, a passive model of arthritis that is independent of adaptive immunity, SFN treatment reduced arthritis severity and synovial hyperplasia. In summary, NQO1 induction by SFN was found to inhibit synoviocyte proliferation and TH17 cell activation in vitro. Moreover, the anti-arthritic and immune regulatory effects of SFN were confirmed in vivo, which suggests that SFN may offer a possible treatment option for RA.

Sulforaphane Inhibits Prostate Carcinogenesis and Pulmonary Metastasis in TRAMP Mice in Association with Increased Cytotoxicity of Natural Killer Cells

The present study shows that oral gavage of 6 mumol d,l-sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived L isomer, thrice per week beginning at 6 weeks of age, significantly inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice without causing any side effects. The incidence of the prostatic intraepithelial neoplasia and well-differentiated (WD) carcinoma were approximately 23% to 28% lower (P < 0.05 compared with control by Mann-Whitney test) in the dorsolateral prostate (DLP) of SFN-treated mice compared with controls, which was not due to the suppression of T-antigen expression. The area occupied by the WD carcinoma was also approximately 44% lower in the DLP of SFN-treated mice relative to that of control mice (P = 0.0011 by Mann Whitney test). Strikingly, the SFN-treated mice exhibited approximately 50% and 63% decrease, respectively, in pulmonary metastasis incidence and multiplicity compared with control mice (P < 0.05 by t test). The DLP from SFN-treated mice showed decreased cellular proliferation and increased apoptosis when compared with that from control mice. Additionally, SFN administration enhanced cytotoxicity of cocultures of natural killer (NK) cells and dendritic cells (DC) against TRAMP-C1 target cells, which correlated with infiltration of T cells in the neoplastic lesions and increased levels of interleukin-12 production by the DC. In conclusion, the results of the present study indicate that SFN administration inhibits prostate cancer progression and pulmonary metastasis in TRAMP mice by reducing cell proliferation and augmenting NK cell lytic activity.