Benzyl Isothiocyanate Treatment Research Articles

Abstract 224: The role of Bmi-1 in benzyl isothiocyanate-mediated suppression of breast cancer stem cell self-renewal

Abstract We have shown previously that benzyl isothiocyanate (BITC), a nontoxic small-molecule constituent of edible cruciferous vegetables (eg, garden cress) inhibits breast cancer stem cells (bCSC) in vitro and in vivo but the underlying mechanisms are not fully understood. Bmi-1 is a member of the polycomb group family of proteins that function in the epigenetic silencing of genes governing self-renewal, differentiation, and proliferation. The present study was undertaken to investigate the role of Bmi-1 in BITC-mediated inhibition of bCSC. BITC treatment downregulated protein level of Bmi-1 in cultured human breast cancer cells (MCF-7, SUM159 and MDA-MB-231), and in MDA-MB-231 xenografts in vivo. Ectopic expression of Bmi-1 alone was sufficient to drive stemness in MCF-7 cells. In addition, Bmi-1 overexpression in MCF-7 cells conferred marked protection against BITC-mediated inhibition of bCSC as evidenced by mammosphere frequency and aldehyde dehydrogenase 1 (ALDH1) activity. Consistent with these observations, silencing of Bmi-1 in SUM159 cells remarkably reduced the portion of bCSC. Apoptosis induced by BITC was also partially attenuated by overexpression of Bmi-1 in MCF-7 cells but its silencing had no effect on BITC-induced apoptosis in SUM159 cells. Previous studies have shown that miRNA128 inhibits cell proliferation and self-renewal by direct regulation of Bmi-1. BITC treatment modestly but statistically significantly increased the expression of miRNA128 in both MCF-7 and SUM159 cells. Inhibition of miRNA128 increased the mammosphere formation in MCF-7 and SUM159 cells and partially attenuated BITC-mediated inhibition of mammosphere formation. These observations led us to conclude that Bmi-1 is a novel target of BITC-mediated inhibition of bCSC. This study was supported by the grant RO1 CA129347 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Shivendra V. Singh. The role of Bmi-1 in benzyl isothiocyanate-mediated suppression of breast cancer stem cell self-renewal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 224. doi:10.1158/1538-7445.AM2014-224

Abstract 225: Overexpression of truncated Ron augments benzyl isothiocyanate-induced apoptosis in human breast cancer cells

Abstract Phytochemicals from edible plants remain attractive for prevention of breast cancer, which is a leading cause of cancer-related mortality in women worldwide. We have shown previously that benzyl isothiocyanate (BITC), a small-molecule found in edible cruciferous vegetables such as garden cress, confers significant protection against mammary cancer development in MMTV-neu transgenic mice. BITC-mediated prevention of mammary carcinogenesis in MMTV-neu mice in vivo is associated with tumor cell apoptosis induction and elimination of breast cancer stem cells (bCSC). In cultured human breast cancer cells, BITC-induced apoptosis is accompanied by activation of multidomain proapoptotic protein Bax whereas inhibition of bCSC self-renewal is significantly attenuated by overexpression of truncated Ron (recepteur d’origine nantais) tyrosine kinase. Ron expression is very low in normal human breast epithelium but its overexpression has been reported in about 50% of primary breast cancers. In addition, mammary-specific overexpression of wild-type Ron as well as its constitutively active form induces highly metastatic breast tumors in mice. The present study was undertaken to study the role of truncated Ron (sfRon) in BITC-induced apoptosis using MCF-7 and MDA-MB-361 human breast cancer cells. Overexpression of sfRon resulted in statistically significant augmentation of BITC-induced apoptotic cell death in both cell lines as judged by flow cytometry after staining with Annexin V and propidium iodide, analysis of histone associated DNA fragment release into the cytosol, and/or cell proliferation assay. On the other hand, sfRon overexpressing MCF-7 cells were resistant to BITC-mediated G2/M phase cell cycle arrest when compared with parental cells. Increased apoptosis following BITC treatment in sfRon overexpressing cells was due to enhanced activation of Bax and/or Bak. Taken together, these findings indicate that overexpression of sfRon sensitizes human breast cancer cells to BITC-induced apoptosis. This investigation was supported by the NCI grant CA129347-06. Citation Format: Anuradha Sehrawat, Shivendra V. Singh. Overexpression of truncated Ron augments benzyl isothiocyanate-induced apoptosis in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 225. doi:10.1158/1538-7445.AM2014-225

Insights into the mode of action of benzyl isothiocyanate on Campylobacter jejuni.

Campylobacter jejuni is a widespread pathogen responsible for most of the food-borne gastrointestinal diseases in Europe. The use of natural antimicrobial molecules is a promising alternative to antibiotic treatments for pathogen control in the food industry. Isothiocyanates are natural antimicrobial compounds, which also display anticancer activity. Several studies described the chemoprotective effect of isothiocyanates on eukaryotic cells, but the antimicrobial mechanism is still poorly understood. We investigated the early cellular response of C. jejuni to benzyl isothiocyanate by both transcriptomic and physiological approaches. The transcriptomic response of C. jejuni to benzyl isothiocyanate showed upregulation of heat shock response genes and an impact on energy metabolism. Oxygen consumption was progressively impaired by benzyl isothiocyanate treatment, as revealed by high-resolution respirometry, while the ATP content increased soon after benzyl isothiocyanate exposition, which suggests a shift in the energy metabolism balance. Finally, benzyl isothiocyanate induced intracellular protein aggregation. These results indicate that benzyl isothiocyanate affects C. jejuni by targeting proteins, resulting in the disruption of major metabolic processes and eventually leading to cell death.

PP141: Benzyl isothiocyanate enhances chemothrerapy and reduces migration and invasion of head and neck squamous cell carcinoma cells

Purpose Approximately 500,000 cases of head and neck squamous cell carcinoma (HNSCC) are reported worldwide each year. Despite recent improvements in cancer treatment, the increase in overall survival of advanced HNSCC has not improved in the past three decades. Consequently, our objective is to find new therapeutic options to enhance survival of patients with aggressive HNSCC. Benzyl isothiocyanate (BITC), a natural compound found in cruciferous vegetables, is showing promising results in targeting chemoresistant and metastatic HNSCC cells. Materials and methods Cell viability after BITC, cisplatin, and BITC followed by cisplatin treatment was determined using a standard MTT assay. Changes in HNSCC migration were investigated using a wound-healing assay. Boyden Chambers were used to study changes in invasion after BITC treatment of HNSCC cells. Immunofluorescence and Western blots were used to determine changes in expression of vimentin and ALDH1. Results Our current data suggests that BITC enhances sensitivity of HNSCC cell lines to cisplatin after 24, 48, and 72 h. A migration assay indicated that the wound healing of metastatic HNSCC cell lines are inhibited by BITC in a dose dependent manner. Additionally, an invasion assay showed that BITC significantly inhibits HNSCC cell invasion towards the chemoattractant EGF after 20 h, in a dose dependent manner. Immunofluorescence and Western blot analysis suggest that BITC inhibits the expression of epithelial–mesenchymal transition marker, vimentin, and putative cancer stem cell marker ALDH1 in the HN12 cell line. Conclusions The present results suggest that BITC may be able to enhance the effect of chemotherapy, while also reducing migration and invasion of HNSCC cells. Thus suggesting that BITC may be a novel adjuvant therapy for patients with aggressive HNSCC.

Abstract 3687: Benzyl isothiocyanate downregulates Ronβ and sfRon to inhibit self-renewal of breast cancer stem cells.

Abstract Recent studies suggest that cancer stem cells play a major role not only in the initiation and maintenance of breast cancer but also in clinical response to therapy. Breast cancer stem cells (bCSC) are characterized by their ability to self-renew (asymmetrical division into a daughter bCSC and a progeny), form spheres under ultra-low attachment conditions in vitro, and express unique cell-surface markers (eg, CD44, ESA). Elimination of both therapy-resistant bCSC and therapy-sensitive epithelial tumor cells may be required for effective prevention of breast cancers. We have shown previously that dietary administration of benzyl isothiocyanate (BITC), a small molecule constituent of many edible cruciferous vegetables (eg, garden cress), significantly inhibits mammary cancer development in a transgenic mouse model (MMTV-neu) in association with apoptosis induction and tumor infiltration of T cells. The present study was undertaken to determine the effect of BITC on bCSC. Exposure of MCF-7 (an estrogen-responsive cell line) and SUM159 (a triple-negative cell line) human breast cancer cells to pharmacologic doses of BITC (0.5-2.5 μM) resulted in inhibition of mammosphere formation (first generation after BITC treatment and subsequent generations without BITC treatment), aldehyde dehydrogenase 1 (ALDH1) activity, and CD44high/CD24low/ESA+ population. Inhibition of bCSC self-renewal resulting from BITC exposure was accompanied by down-regulation of urokinase-type plasminogen activator (uPAR), Bmi-1, and Ron (Ronβ and sfRon) proteins in vitro and in MDA-MB-231 xenografts in vivo (uPAR and Bmi-1). Ectopic expression of uPAR alone was sufficient to drive stemness in MCF-7 and MDA-MB-468 cells. However, the BITC-mediated inhibition of mammosphere formation and ALDH1 activity was sustained even after stable overexpression of uPAR in MCF-7 or MDA-MB-468 cells. Similarly, Bmi-1 overexpression was largely dispensable for BITC-mediated suppression of bCSC. On the other hand, overexpression of Ron (full-length) as we as sfRon in MCF-7 cells conferred significant protection against BITC-mediated inhibition of bCSC self-renewal. Ectopic expression of sfRon, but not Ron, resulted in nearly 4-fold increase in ALDH1 activity compared with control cells. Novel conclusions of the present study are: (a) overexpression of sfRon is sufficient to drive stemness at least in MCF-7 cells (b) mammary cancer prevention by BITC likely involves removal of bCSC, and (c) BITC-mediated inhibition of bCSC is caused by suppression of Ron signaling. This study was supported by the grant CA129347-06 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Anuradha Sehrawat, Shivendra V. Singh. Benzyl isothiocyanate downregulates Ronβ and sfRon to inhibit self-renewal of breast cancer stem cells. [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 3687. doi:10.1158/1538-7445.AM2013-3687

Abstract 3671: The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells.

Abstract We have shown previously that breast cancer chemoprevention by benzyl isothiocyanate (BITC) in mouse mammary tumor virus-neu transgenic mice is associated with induction of E-cadherin protein expression. Loss of E-cadherin expression concomitant with induction of mesenchymal markers (eg, vimentin and fibronectin) is a biochemical hallmark of epithelial-to-mesenchymal transition (EMT). EMT is a developmental process but implicated in progression of cancer to aggressive state. We have also shown previously that BITC treatment inhibits EMT in cultured MDA-MB-231 human breast cancer cells. The present study extends these observations and investigates the mechanism by which BITC inhibits EMT. Initially, we designed experiments using another triple-negative human breast cancer cell line (SUM159) to determine generality of the BITC-mediated inhibition of EMT. Exposure of SUM159 cells to pharmacologically-relevant concentrations of BITC (2.5 and 5 μM) resulted in induction of E-cadherin protein expression, which was accompanied by suppression of vimentin protein level especially at the 5 μM dose. Protein levels of slug and snail, which are transcriptional repressors of E-cadherin, were also decreased markedly after treatment of SUM159 cells with BITC, but this effect was relatively more pronounced on slug protein level compared with snail. Similar to MDA-MB-231 cells, the BITC-mediated induction of E-cadherin protein expression in SUM159 cells was accompanied by its transcriptional upregulation as revealed by E-cadherin luciferase reporter assay. Immunofluorescence microscopy confirmed induction of E-cadherin protein and suppression of vimentin protein expression in BITC-treated SUM159 cells. BITC treatment resulted in downregulation of Forkhead Box Q1 transcription factor (FOXQ1) protein and FOXQ1 mRNA in MDA-MB-231, SUM159, and MDA-MB-468 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with suppression of FOXQ1 protein level. Ectopic expression of FOXQ1 in a mammary epithelial cell line conferred marked protection against BITC-mediated inhibition of EMT as well as cell migration. In conclusion, the present study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells. This study was supported by the grant CA129347-06 awarded by the National Cancer Institute. Citation Format: Anuradha Sehrawat, Su-Hyeong Kim, Andreas Vogt, Shivendra V. Singh. The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells. [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 3671. doi:10.1158/1538-7445.AM2013-3671

Suppression of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells

We showed previously that breast cancer chemoprevention with benzyl isothiocyanate (BITC) in MMTV-neu mice was associated with induction of E-cadherin protein in vivo. Loss of E-cadherin expression and induction of mesenchymal markers (e.g. vimentin) are biochemical hallmarks of epithelial-mesenchymal transition (EMT), a developmental process implicated in progression of cancer to aggressive state. This study offers novel insights into the mechanism by which BITC inhibits EMT. Exposure of MDA-MB-231, SUM159 and MDA-MB-468 human breast cancer cells to BITC (2.5 and 5 µM) resulted in transcriptional repression of urokinase-type plasminogen activator (uPA) as well as its receptor (uPAR). However, ectopic expression of uPAR in MDA-MB-468 cells failed to confer protection against induction of E-cadherin and inhibition of cell invasion/migration resulting from BITC treatment. The BITC-mediated induction of E-cadherin and inhibition of cell migration was sustained in MDA-MB-231 and SUM159 cells transiently transfected with an uPAR-targeted small interfering RNA. Overexpression of Forkhead Box Q1 (FOXQ1), whose protein and messenger RNA levels were decreased by BITC treatment in cells and MDA-MB-231 xenografts, conferred marked protection against BITC-mediated inhibition of EMT and cell migration. In conclusion, this study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells.

Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling

Benzyl isothiocyanate (BITC) is a dietary chemopreventive agent that inhibits the growth of various human cancer cells by causing apoptotic cell death. In this study, we demonstrate that BITC not only induces apoptosis but also induces autophagy in human hormone-sensitive (Rv1) and -refractory (PC3) prostate cancer cells. In BITC-treated cells, the induction of autophagy was detected by monitoring the processing of an autophagy marker protein, microtubule-associated protein 1 light chain 3 (LC3), the aggregation of LC3 into granular structures and the formation of acidic organelles. Inhibition of autophagy using 3-methyladenine increased BITC-induced apoptosis, whereas the administration of caspase inhibitor suppressed BITC-induced cell death. Our data also showed that BITC inhibits mammalian target of rapamycin (mTOR) kinase activity in a dose-dependent manner. The expression of phospho-mTOR (Ser2481), an indicator of mTOR intrinsic catalytic activity, and phospho-UNC-51-like kinase 1 (Ser757), a direct substrate of mTOR, were decreased in BITC-treated cells. However, the increased expression of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and antiapoptotic Bcl-2 were detected only in PC3 cells at later stages of BITC treatment. Collectively, our results show that BITC induces a protective autophagy response in Rv1 and PC3 cells through inhibition of the mTOR signaling pathway. Activation of the AKT survival pathway was only observed in PC3 cells, representing a resistance mechanism of advanced prostate cancer upon BITC treatment. These findings could potentially contribute to the beneficial effect of BITC in prostate cancer treatments.

Abstract 565: Benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells is associated with downregulation of urokinase-type plasminogen activator and its receptor

Abstract Novel approaches for chemoprevention of breast cancer are clinically attractive not only because many risk factors associated with this disease are not easily modifiable (e.g., genetic predisposition) but also due to the fact that the currently available chemopreventive options targeted against breast cancer (e.g., estrogen receptor antagonists) are sub-optimal and plagued with adverse side effects. Needless to emphasize that breast cancer continues to be a leading cause of cancer-related death in American women despite significant advances towards targeted therapies. Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., garden cress), seems promising for chemoprevention of breast cancer based on our own preclinical work. For example, we have shown previously that BITC administration not only inhibits growth of human breast cancer xenografts (MDA-MB-231) in athymic mice but also imparts significant protection against mammary cancer development in a transgenic mouse model (MMTV-neu). More recent studies from our laboratory have revealed that BITC is a potent inhibitor of epithelial-mesenchymal transition (EMT) in human breast cancer cells, but the mechanism underlying this effect is not known. In the present study, we addressed this question using MDA-MB-231, MDA-MB-468, and SUM159 human breast cancer cells as a model. Overexpression of superoxide dismutase, which confers significant protection against proapoptotic and proautophagic cell death responses to BITC, did not have any meaningful impact on BITC-mediated inhibition of EMT. Likewise, ectopic expression of constitutively active STAT3, a transcription factor implicated in EMT regulation, had no effect on EMT inhibition by BITC. Instead, the BITC-mediated inhibition of EMT was associated with suppression of protein and mRNA levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). Secretion of uPA in culture media of breast cancer cells was also decreased significantly upon BITC treatment. BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was accompanied by downregulation of uPA and uPAR protein expression in the tumor and suppression of uPA secretion in plasma. Ectopic expression of uPAR in MDA-MB-468 and MCF-7 cells conferred partial but significant protection against BITC-mediated inhibition of EMT, cell migration, and/or cell invasion. Collectively, these results indicate that BITC-mediated inhibition of EMT is caused, at least in part, by suppression of the uPA/uPAR system. This investigation was supported by the USPHS grant RO1 CA129347-05, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 565. doi:1538-7445.AM2012-565

Notch2 activation by benzyl isothiocyanate impedes its inhibitory effect on breast cancer cell migration

Benzyl isothiocyanate (BITC) is a promising anticancer constituent of edible cruciferous vegetables with in vivo efficacy against chemically induced as well as oncogene-driven breast cancer in experimental rodents. However, the mechanism underlying anticancer effect of BITC is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer responses to BITC as this pathway is often hyperactive in human breast cancer. Exposure of MCF-7, MDA-MB-231, and SUM159 human breast cancer cells to pharmacologic concentrations of BITC (2.5 and 5 μM) resulted in cleavage (activation) of Notch1, Notch2, and Notch4, which was accompanied by induction of γ-secretase complex components Presenilin1 and/or Nicastrin. The BITC-mediated cleavage of Notch was associated with its transcriptional activation as revealed by RBP-Jk and Hes-1A/B luciferase reporter assays. Inhibition of cell migration or cell viability resulting from BITC exposure was not influenced by pharmacological suppression of Notch1 using a γ-secretase inhibitor or RNA interference of Notch1 as well as Notch4. On the other hand, the BITC-mediated inhibition of cell migration, but not cell viability, was significantly augmented by siRNA and shRNA knockdown of Notch2 protein. Furthermore, the BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with a significant increase in nuclear levels of cleaved Notch2 and Hes-1 proteins. In conclusion, the results of this study indicate that (a) BITC treatment activates Notch2 in cultured and xenografted human breast cancer cells, and (b) Notch2 activation impedes inhibitory effect of BITC on cell migration.

Benzyl Isothiocyanate Causes FoxO1-Mediated Autophagic Death in Human Breast Cancer Cells

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.

Critical Role of p53 Upregulated Modulator of Apoptosis in Benzyl Isothiocyanate-Induced Apoptotic Cell Death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.

Benzyl Isothiocyanate (BITC) Induces G2/M Phase Arrest and Apoptosis in Human Melanoma A375.S2 Cells through Reactive Oxygen Species (ROS) and both Mitochondria-Dependent and Death Receptor-Mediated Multiple Signaling Pathways

Benzyl isothiocyanates (BITC), a member of the isothiocyanate (ITC) family, inhibits cell growth and induces apoptosis in many types of human cancer cell lines. The present study investigated mechanisms underlying BITC-induced apoptosis in A375.S2 human melanoma cancer cells. To observe cell morphological changes and viability, flow cytometric assays, cell counting, and a contrast-phase microscopic examination were carried out in A375.S2 cells after BITC treatment. Cell cycle distribution and apoptosis were assessed with the analysis of cell cycle by flow cytometric assays, DAPI staining, propidium iodide (PI), and annexin V staining. Apoptosis-associated factors such as reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and caspase-3 activity were evaluated by flow cytometric assays. Abundance of cell cycle and apoptosis associated proteins was determined by Western blotting. AIF and Endo G expression was examined by confocal laser microscope. Results indicated that (1) BITC significantly reduced cell number and induced cell morphological changes in a dose-dependent manner in A375.S2 cells; (2) BITC induced arrest in cell cycle progression at G(2)/M phase through cyclin A, CDK1, CDC25C/Wee1-mediated pathways; (3) BITC induced apoptosis and increased sub-G(1) population; and (4) BITC promoted the production of ROS and Ca(2+) and loss of ΔΨ(m) and caspase-3 activity. Furthermore, BITC induced the down-regulation of Bcl-2 expression and induced up-regulation of Bax in A375.S2 cells. Moreover, BITC-induced cell death was decreased after pretreatment with N-acetyl-l-cysteine (NAC, a ROS scavenger) in A375.S2 cells. In conclusion, the results showed that BITC promoted the induction of G(2)/M phase arrest and apoptosis in A375.S2 human melanoma cells through ER stress- and mitochondria-dependent and death receptor-mediated multiple signaling pathways. These data suggest that BITC has potential as an agent for the treatment of melanoma.

Benzyl Isothiocyanate Suppresses Pancreatic Tumor Angiogenesis and Invasion by Inhibiting HIF-α/VEGF/Rho-GTPases: Pivotal Role of STAT-3

Our previous studies have shown that benzyl isothiocyanate (BITC) suppresses pancreatic tumor growth by inhibiting STAT-3; however, the exact mechanism of tumor growth suppression was not clear. Here we evaluated the effects and mechanism of BITC on pancreatic tumor angiogenesis. Our results reveal that BITC significantly inhibits neovasularization on rat aorta and Chicken-Chorioallantoic membrane. Furthermore, BITC blocks the migration and invasion of BxPC-3 and PanC-1 pancreatic cancer cells in a dose dependant manner. Moreover, secretion of VEGF and MMP-2 in normoxic and hypoxic BxPC-3 and PanC-1 cells was significantly suppressed by BITC. Both VEGF and MMP-2 play a critical role in angiogenesis and metastasis. Our results reveal that BITC significantly suppresses the phosphorylation of VEGFR-2 (Tyr-1175), and expression of HIF-α. Rho-GTPases, which are regulated by VEGF play a crucial role in pancreatic cancer progression. BITC treatment reduced the expression of RhoC whereas up-regulated the expression of tumor suppressor RhoB. STAT-3 over-expression or IL-6 treatment significantly induced HIF-1α and VEGF expression; however, BITC substantially suppressed STAT-3 as well as STAT-3-induced HIF-1α and VEGF expression. Finally, in vivo tumor growth and matrigel-plug assay show reduced tumor growth and substantial reduction of hemoglobin content in the matrigel plugs and tumors of mice treated orally with 12 µmol BITC, indicating reduced tumor angiogenesis. Immunoblotting of BITC treated tumors show reduced expression of STAT-3 phosphorylation (Tyr-705), HIF-α, VEGFR-2, VEGF, MMP-2, CD31 and RhoC. Taken together, our results suggest that BITC suppresses pancreatic tumor growth by inhibiting tumor angiogenesis through STAT-3-dependant pathway.

Benzyl isothiocyanate sensitizes human pancreatic cancer cells to radiation by inducing apoptosis

Isothiocyanates are a class of naturally occurring chemopreventive agents known to suppress proliferation of cancer cells in culture. The present study was undertaken in order to examine the effects of benzyl isothiocyanate (BITC), one of the common dietary isothiocyanates, on the radiosensitivity of human pancreatic cancer cells and to gain insights into the underlying molecular mechanism of BITC-induced radiosensitization. Two human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2, were treated with BITC and irradiated with X-rays. Radiation sensitivity, apoptosis, and protein levels were determined by a clonogenic assay, fluorescence microscopic analysis with DAPI staining and Western blotting, respectively. MIAPaCa-2 cells were relatively more sensitive to BITC treatment compared with PANC-1 cells. Radiosensitization was observed in both PANC-1 and MIAPaCa-2 cells incubated with BITC at 5 to 10 µM and 2.5 to 5 µM for 24 h, respectively. The combination treatments with BITC and X-rays also revealed an increased percentage of apoptotic cells. In addition, treatment with BITC and X-rays resulted in a decrease in the protein levels of the X-linked inhibitor of apoptosis (XIAP), inhibitor of apoptosis (IAP) family protein, and in a marked increase in the apoptosis protease activating factor-1 (Apaf-1), essential for activation of caspase-9 in stress-induced apoptosis. BITC may be a useful radiosensitizer for radiotherapy of pancreatic cancers.

Benzyl Isothiocyanate Inhibits Epithelial-Mesenchymal Transition in Cultured and Xenografted Human Breast Cancer Cells

We showed previously that cruciferous vegetable constituent benzyl isothiocyanate (BITC) inhibits growth of cultured and xenografted human breast cancer cells and suppresses mammary cancer development in a transgenic mouse model. We now show, for the first time, that BITC inhibits epithelial-mesenchymal transition (EMT) in human breast cancer cells. Exposure of estrogen-independent MDA-MB-231 and estrogen-responsive MCF-7 human breast cancer cell lines and a pancreatic cancer cell line (PL-45) to BITC resulted in upregulation of epithelial markers (e.g., E-cadherin and/or occludin) with a concomitant decrease in protein levels of mesenchymal markers, including vimentin, fibronectin, snail, and/or c-Met. The BITC-mediated induction of E-cadherin protein was accompanied by an increase in its transcription, whereas BITC-treated MDA-MB-231 cells exhibited suppression of vimentin, snail, and slug mRNA levels. Experimental EMT induced by exposure to TGFβ and TNFα or Rb knockdown in a spontaneously immortalized nontumorigenic human mammary epithelial cell line (MCF-10A) was also partially reversed by BITC treatment. The TGFβ-/TNFα-induced migration of MCF-10A cells was inhibited in the presence of BITC, which was partially attenuated by RNA interference of E-cadherin. Inhibition of MDA-MB-231 xenograft growth in vivo in female athymic mice by BITC administration was associated with an increase in protein level of E-cadherin and suppression of vimentin and fibronectin protein expression. In conclusion, this study reports a novel anticancer effect of BITC involving inhibition of EMT, a process triggered during progression of cancer to invasive state.

Abstract 860: Benzyl isothiocyanate inhibits oncogenic actions of leptin in human breast cancer cells by suppressing activation of signal transducer and activator of transcription 3

Abstract Obesity is a well-established risk factor for breast cancer progression and molecular effects of obesity are mediated by adipocytokine, leptin. Considering the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., gardencress), confers significant protection against mammary carcinogenesis in a transgenic mouse model (MMTV-neu). The BITC-mediated prevention of mammary cancer development in MMTV-neu mice correlated with reduced cell proliferation, increased apoptosis, and tumor infiltration of T cells. The present study provides first evidence for efficacy of BITC against oncogenic effects of letpin. BITC-treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 human breast cancer cells (an estrogen-responsive cell line with wild-type p53) at pharmacologic doses. Leptin-stimulated migration and invasion of these cells was also inhibited significantly in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC-treatment suppressed leptin-induced phosphorylation of signal transducer and activator of transcription 3 (Stat3) and cyclin D1 transactivation. BITC-treatment efficiently inhibited Stat3 recruitment to the cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a decrease in level of phospho-Stat3. These studies indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivastion. Taken together, these data show that BITC treatment can inhibit the oncogenic actions of leptin via blocking its downstream signaling molecule, Stat3. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. This work was supported in part by the USPHS grants NCI-RO1 CA129347 (to SVS), NIDDK-K01DK076742 and R03DK089130 (to NKS), NCI-R01CA131294 and NCI-R21 CA155686 (to DS). 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 860. doi:10.1158/1538-7445.AM2011-860

Pancreatic Tumor Suppression by Benzyl Isothiocyanate Is Associated with Inhibition of PI3K/AKT/FOXO Pathway

Our previous studies have shown that benzyl isothiocyanate (BITC) suppress pancreatic cancer growth by inducing apoptosis but the molecular mechanism was unclear. In this study we hypothesized the involvement of PI3K/AKT/FOXO pathway in BITC-induced apoptosis. Mice were implanted BxPC-3 tumor xenografts and orally gavaged with 12 μmol BITC. Plasma and tumor BITC concentration was estimated by liquid chromatography/tandem mass spectrometry. BxPC-3 and PanC-1 cells were used to elucidate PI3K/AKT/FOXO pathway. Electrophoretic mobility shift assay (EMSA), DNA binding activity, immunofluorescence, and gene transfection were used to delineate the mechanism. BITC-treated mice showed 43% less tumor growth as compared with control mice and correlated well with the therapeutic concentrations of 6.5 μmol/L BITC achieved in plasma and 7.5 μmol/g BITC in tumor tissue. Western blot analyses and immunohistochemistry revealed that tumors from BITC-treated mice showed reduced phosphorylation of PI3K, AKT, PDK1, mTOR, FOXO1, and FOXO3a and increased apoptosis. Complementing our in vivo results, we made similar observations in a dose- and time-dependent manner in BITC-treated BxPC-3 and Panc-1 cells. Binding of FOXO1 with 14-3-3 proteins was also reduced drastically by BITC treatment indicating nuclear retention of FOXO1 and this observation was further confirmed with EMSA, immunofluorescence, DNA binding, and upregulation of FOXO-responsive proteins Bim, p27, and p21 in BxPC-3 cells. Overexpression of AKT by transient transfection significantly blocked the modulation of FOXO proteins and protected the cells from BITC-mediated apoptosis and growth suppression. Our results provide convincing evidence on the involvement of PI3K/AKT/FOXO pathway in BITC-mediated pancreatic tumor growth suppression.

Oral administration of benzyl-isothiocyanate inhibits solid tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells in BALB/c mice

Benzyl-isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has also been shown to have anti-tumor properties. To evaluate the effects of BITC administration on the tumor growth and metastasis of breast cancer, 4T1 murine mammary carcinoma cells were injected into the inguinal mammary fat pads of syngeneic female BALB/c mice. One day later, the mice were subjected to gavage for 4weeks with BITC (0, 5, or 10mg/kg body weight/day). Oral BITC treatment induced a significant reduction in the growth of solid tumors. BITC reduced hemoglobin contents and CD31 and vascular endothelial growth factor (VEGF) expression in the tumors, as well as circulating levels of VEGF. Reduced expressions of proliferating cell nuclear antigen and cyclin-dependent kinase 4 were noted in the tumors of BITC-treated mice. BITC markedly increased the numbers of apoptotic cells with increased Bax expression, cleaved caspase-3, and PARP levels, but reduced Bcl-2 expression in tumor tissues. In addition, BITC was shown to reduce the numbers of pulmonary tumor nodules and the total pulmonary metastatic volume. BITC induced a significant reduction in the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and urokinase-type plasminogen activator in the sera and lungs of 4T1 cell-injected mice. However, the concentrations of TIMP-2 and plasminogen activator inhibitor-1 were increased in the sera and lungs of BITC-treated mice. The results of this study indicate that BITC has potential as a preventive agent for metastatic breast cancer.

Benzyl isothiocyanate inhibits oncogenic actions of leptin in human breast cancer cells by suppressing activation of signal transducer and activator of transcription 3

Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g. garden cress), confers significant protection against mammary carcinogenesis in a transgenic mouse model. The present study provides first evidence for the efficacy of BITC against oncogenic effects of leptin. The BITC treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 and MCF-7 human breast cancer cells. Leptin-stimulated migration and invasion of these cells was also inhibited in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC treatment suppressed leptin-induced Stat3 phosphorylation and cyclin D1 transactivation. The BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a modest yet significant decrease in levels of Tyr705 phosphorylated Stat3. The BITC treatment efficiently inhibited Stat3 and SRC1 recruitment to cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore, overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation, whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. These results indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivation. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.