Effects Of Benzyl Isothiocyanate Research Articles

Antimalarial Activity of Aqueous Extracts of Nasturtium (Tropaeolum majus L.) and Benzyl Isothiocyanate.

Malaria remains an important and challenging infectious disease, and novel antimalarials are required. Benzyl isothiocyanate (BITC), the main breakdown product of benzyl glucosinolate, is present in all parts of Tropaeolum majus L. (T. majus) and has antibacterial and antiparasitic activities. To our knowledge, there is no information on the effects of BITC against malaria. The present study evaluates the antimalarial activity of aqueous extracts of BITC and T. majus seeds, leaves, and stems. We used flow cytometry to calculate the growth inhibition (GI) percentage of the extracts and BITC against unsynchronized cultures of the chloroquine-susceptible Plasmodium falciparum 3D7 - GFP strain. Extracts and/or compounds with at least 70% GI were validated by IC50 estimation against P. falciparum 3D7 - GFP and Dd2 (chloroquine-resistant strain) unsynchronized cultures by flow cytometry, and the resistance index (RI) was determined. T. majus aqueous extracts showed some antimalarial activity that was higher in seeds than in leaves or stems. BITC's GI was comparable to chloroquine's. BITC's IC50 was similar in both strains; thus, a cross-resistance absence with aminoquinolines was found (RI < 1). BITC presented features that could open new avenues for malaria drug discovery.

Bacteriostatic effects of benzyl isothiocyanate on Vibrio parahaemolyticus: Transcriptomic analysis and morphological verification

BackgroundFoodborne illness caused by Vibrio parahaemolyticus (V. parahaemolyticus) is generally associated with the consumption of seafood. Fish and other seafood can be contaminated with V. parahaemolyticus, natural inhabitants of the marine, estuarine, and freshwater environment. In this study, the antibacterial activities of benzyl isothiocyanate (BITC) against V. parahaemolyticus were investigated by both transcriptomic analysis and morphological verification.ResultsTreatment with 1/8 minimum inhibitory concentration (1/8 MIC) BITC resulted in 234 upregulated genes and 273 downregulated genes. The results validated by quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the relative expression levels of the six genes VP0820, VP0548, VP2233, VPA2362, fliA and fliG were only 31.0%, 31.1%, 55.8%, 57.0%, 75.3%, and 79.9% of the control group, respectively. Among them, genes VP2233, fliA and fliG are related to flagella and VP2362 can regulate a protein relevant to biofilm formation. Morphologically, we verified that the swimming diffusion diameter of V. parahaemolyticus was significantly reduced by 14.9% by bacterial swimming ability, and biofilm formation was significantly inhibited by treatment with 1/8 MIC BITC by crystal violet quantification assay.ConclusionsThese results indicated that 1/8 MIC BITC had antibacterial effect on V. parahaemolyticus by inhibiting virulence gene expression related to flagella and biofilm.

Benzyl Isothiocyanate Induces Apoptosis and Inhibits Tumor Growth in Canine Mammary Carcinoma via Downregulation of the Cyclin B1/Cdk1 Pathway.

Background: Canine mammary carcinoma is common in female dogs, and its poor prognosis remains a serious clinical challenge, especially in developing countries. Benzyl isothiocyanate (BITC) has attracted great interest because of its inhibitory effect against tumor activity. However, its effect and the underlying mechanisms of action in canine mammary cancer are not well-understood. Here, we show that BITC suppresses mammary tumor growth, both in vivo and in vitro, and reveal some of the potential mechanisms involved.Methods: The effect of BITC on canine mammary cancer was evaluated on CIPp and CMT-7364, canine mammary carcinoma lines. The cell lines were treated with BITC and then subjected to wound healing and invasion assays. Cell cycles and apoptosis were measured using flow cytometry; TUNEL assay; immunohistochemistry (IHC) for caspase 3, caspase 9, and cyclin D1; hematoxylin and eosin (H&E) staining; and/or quantitative polymerase chain reaction (qPCR).Results: BITC showed a strong suppressive effect in both CIPp and CMT-7364 cells by inhibiting cell growth in vitro; these effects were both dose- and time-dependent. BITC also inhibited migration and invasion of CIPp and CMT-7364 cells. BITC induced G2 arrest and apoptosis, decreasing tumor growth in nude mice by downregulation of cyclin B1 and Cdk1 expression.Conclusion: BITC suppressed both invasion and migration of CIPp and CMT-7364 cells and induced apoptosis. BITC inhibited canine mammary tumor growth by suppressing cyclinB1 and Cdk1 expression in nude mice.

The effect of benzyl isothiocyanate on Candida albicans growth, cell size, morphogenesis, and ultrastructure

Candida albicans is a commensal yeast that may become pathogenic and even lethal to the host. Over the last few decades, antifungal resistance has increased, promoting screening of the antifungal potential of old and new substances. This study investigates the antifungal potential of isothiocyanates (ITCs) against C. albicans oral isolates. A preliminary susceptibility disk diffusion test (DD) was performed using allyl isothiocyanate (AITC), benzyl isothiocynanate (BITC) and phenyl ethyl isothiocyanate (PEITC) at a fixed concentration range (0.001-0.1M). Because C. albicans isolates were more susceptible to BITC and PEITC, their effect on cell size and on germ tube formation (GTF) were tested. The most promising molecule, BITC, was further tested for effects on cell viability, oxidative stress and for ultrastructure. ITCs, especially the aromatic ones, had a significant type-, dose- and isolate-dependent anti-Candida activity. Although BITC and PEITC had similar activity against the yeast cells, BITC had a more pronounced effect on cell size and GTF. Furthermore, BITC appears to induce oxidative stress and promote changes in the cell ultrastructure, interfering with cell wall structure. Our work showed that aromatic ITCs have the potential to effect C. albicans cells in multiple ways, including size, shape and GTF (BITC and PEITC), oxidative stress, and ultrastructure (BITC). Overall, our results suggest that BITC may be effectively used against C. albicans to modulate its growth, and control or suppress its invasive potential.

Benzyl Isothiocyanate Induces Apoptotic Cell Death Through Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

Gefitinib is used to treat patients with lung cancer, but in some patients, the disease becomes gefitinib-resistant. Benzyl isothiocyanate (BITC), found in cruciferous vegetables, has shown anticancer activity in many human cancer cell lines. However, the effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells in vitro have not been investigated. The effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells were investigated in vitro. Flow cytometric assay was used for determining the total viable cell number, apoptotic cell death, the production of reactive oxygen species (ROS) and Ca2+, mitochondriaI membrane potential (Ψm) and caspase-3, -8 and -9 activities. Furthermore, 4', 6-diamidino-2-phenylindole staining was used to examine chromatin condensation in NCI-H460 and NCI-H460/G cells. BITC reduced total viable cell number via the induction of apoptotic cell death, that was also confirmed by annexin V/propidium iodide double staining assay. BITC increased ROS and Ca2+ production, reduced Ψm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting assay also showed that BITC increased expression of cleaved caspase-3 and -9, cytochrome c, BCL2-associated X protein, endonuclease G, poly (ADP-ribose) polymerase, growth arrest and DNA-damage protein 153, caspase-7 and activating transcription factor 6 alpha, but reduced apoptosis-inducing factor and caspase-9, BH3-interacting domain death agonist, calpain 1, glucose-regulated protein 78 and inositol requiring enzyme 1 alpha in NCI-H460/G cells. BITC-induced apoptotic cell death appears to occur via caspase- and mitochondria-dependent pathways in both cell lines.

Abstract 5258: Benzyl isothiocyanate prevents breast cancer-induced bone erosion in vivo

Abstract Bone is one of the metastatic sites for advanced breast cancer. Nearly 70% of breast cancer patients experience metastasis to bone due to enhanced osteoclastogenesis and formation of osteoclasts leading to bone resorption. We have shown previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables such as garden cress, is a potent inhibitor of breast cancer cells in vitro and in vivo. The present study was designed to determine the effect of BITC on breast cancer-induced osteoclastogenesis. Co-culture of murine RAW 264.7 macrophages with human breast cancer cells resulted in genesis of osteoclasts in vitro that was suppressed significantly in the presence of BITC. Runx2, a transcription factor, is abnormally expressed in breast cancer and contributes to osteoclastogenesis by regulating receptor activator of NF-κB ligand (RANKL). BITC treatment downregulated expression of Runx2 in breast cancer cells at the mRNA and protein levels. Osteoclastogenesis was increased in Runx2 overexpressing MDA-MB-231 cells, whereas conditional knockdown of Runx2 in T47D cells attenuated this process. To determine the in vivo efficacy of BITC, MDA-MB-231-Luc cells were injected into the left ventricle of nude mice. BITC was administrated orally at 10 mg per kg body weight. Micro-CT X-ray analysis showed that bone metastases and erosion was decreased by about 50% upon BITC treatment when compared with vehicle control. Cathepsin K and total RANKL levels were lower in the plasma of BITC-treated mice when compared to the control group. Altogether, this study demonstrates, for the first time, that BITC is a potent inhibitor of breast cancer-induced osteoclastogenesis in vivo. This study was supported by the grant RO1 CA129347-09 awarded by the national Cancer Institute. Citation Format: Subrata K. Pore, Shivendra V. Singh. Benzyl isothiocyanate prevents breast cancer-induced bone erosion in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5258. doi:10.1158/1538-7445.AM2017-5258

Effects of benzyl isothiocyanate and papain on equine cyathostome egg hatching

Effects of benzyl isothiocyanate and papain on equine cyathostome egg hatching

Abstract 826: Benzyl isothiocyanate inhibits breast cancer-induced osteoclastogenesis

Abstract Breast cancer is a major public health issue for women worldwide and 70% of the disease preferentially metastasizes to bone resulting in its destruction. Advanced breast cancer releases osteoclastogenic factors to promote bone resorption. As bone metastasis with breast cancer is associated with high mortality, osteoclastogenesis is a potential therapeutic target. We have shown previously that benzyl isothiocyanate (BITC), which is present in edible cruciferous vegetables, inhibits breast cancer development in a transgenic mouse model. The present study was designed to determine effect of BITC on breast cancer-induced osteoclastogenesis. We employed well-established in vitro osteoclast co-culture system of Raw 264.7 murine macrophage cells with human breast cancer cells for this assessment. BITC treatment significantly inhibited the number as well as size of osteoclasts in a dose dependent manner in every co-culture experiment. In addition, BITC downregulated both mRNA and protein levels of macrophage colony stimulating factor (MCSF) and receptor activator of NF-êB ligand (RANKL) which are key factors for formation of osteoclasts. Moreover, receptor activator of NF-êB ligand (RANKL)-induced osteoclast differentiation as well as bone resorption was suppressed in the presence of BITC. On the other hand, BITC induced osteoprotegerin (OPG) acting as a decoy receptor of RANKL and negatively regulated osteoclast formation. Quantitative PCR analysis revealed that mRNA levels of osteoclastogenesis-related cytokines and proteins (IL-1â, IL-6, TNF-á, GM-CSF, OPG, RUNX2) were repressed in BITC-treated breast cancer cells. Taken together, BITC showed inhibitory activity on breast cancer-mediated osteoclastogenesis. This study was supported by the grant CA129347 awarded by the National Cancer Institute. Citation Format: Subrata K. Pore, Anuradha Sehrawat, Shivendra V. Singh. Benzyl isothiocyanate inhibits breast cancer-induced osteoclastogenesis. [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 826.

Effects of Benzyl Isothiocyanate and Its N-Acetylcysteine Conjugate on Induction of Detoxification Enzymes in Hepa1c1c7 Mouse Hepatoma Cells

The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with 0.1~10 μM BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; 0.5 μM and 10 μM NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with 0.1~2.0 μM BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with 1 μM and 2 μM BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with 1 μM and 2 μM NAC-BITC caused 1.6- and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in 0.1~2 μM BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in 0.1 μM NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., 1~2 μM) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents.

The effect of benzyl isothiocyanate and its computer-aided design derivants targeting alkylglycerone phosphate synthase on the inhibition of human glioma U87MG cell line.

Benzyl isothiocyanate (BITC) has been shown to have inhibitory potential for human glioma U87MG cells; however, the effect and mechanism were not fully clear. In the present study, we found that BITC could inhibit U87MG cell proliferation, adhesion, invasion, and vasculogenic mimicry (VM) formation potential and induce oxidative stress, apoptosis, and cell cycle arrest. We also found that the expression of proliferation, invasion, VM oxidative stress, apoptosis, and cell cycle-related gene and the activity of tumor-related signaling pathways, including protein kinase C (PKC) ζ and Akt/nuclear factor-kappa B (NF-κB) pathways, were suppressed by BITC treatment. We also explored the anti-tumor potential of BITC in vivo, and we found that BITC also could regulate the expression of tumor-related gene and angiogenesis in nude mice model. Finally, we optimized the BITC construction targeting alkylglycerone phosphate synthase (AGPS) by computer-aided design, and the derivants also showed anti-tumor potential in vitro.

Benzyl isothiocyanate affects development, hatching and reproduction of the soybean cyst nematode Heterodera glycines

Benzyl isothiocyanate (BITC), prepared in 1% methanol and applied at micro-molar doses, decreased Heterodera glycines infective second-stage juvenile (J2) movement, hatching and reproduction on soybean, Glycine max. Direct exposure of J2 to 30 μM BITC caused an immediate decrease (17%) in J2 movement relative to 1% methanol controls, and within 3 h exposure eliminated &gt;99% of movement. Continuous exposure of H. glycines eggs to 30 μM or 60 μM BITC significantly decreased percentage hatch of J2 measured at 7 days (30 μM, 15.8 ± 2.4%; 60 μM, 7.9 ± 2.2%) relative to controls (44.9 ± 4.1%). Control percentage hatch increased between days 7 and 14 (87.5 ± 2.3%), whereas there was no significant increase in hatch after Day 7 in either treatment group. The effect of BITC on hatch was rapid and persistent. Following 4 h exposure of eggs to either 30 μM or 60 μM BITC and transfer to water, percentage hatch at Day 14 was 51.3 ± 6.3% and 15.6 ± 1.4%, respectively. Each value was significantly less than the control percentage hatch (96.5 ± 0.2%). The same treatment method also resulted in decreased reproduction. The mean number of cysts per plant harvested 5 weeks after inoculation with control eggs (173.0 ± 12.1) was significantly greater than the numbers from plants inoculated with either 30 μM (78.9 ± 8.2) or 60 μM (38.3 ± 4.5) BITC treated eggs. Reduction of hatch and reproduction were strongly correlated, with for both 30 μM or 60 μM BITC treatments. The effects of BITC were primarily on the early embryo. The percent of all embryos in the multicellular stage at Day 14 in control eggs was 4.9 ± 0.7%, but increased to 35.1 ± 4.3% (30 μM) and 56.3 ± 4.5% (60 μM) in BITC treated eggs. BITC has multiple effects on H. glycines, affecting both infective juvenile movement and embryonic development, and decreasing reproduction.

Inhibitory Effect of Benzyl Isothiocyanate on Proliferation in vitro of Human Glioma Cells

Malignant glioma, also known as brain cancer, is the most common intracranial tumor, having an extremely high mortality and recurrence rate. The survival rate of the affected patients is very low and treatment is difficult. Hence, growth inhibition of glioma has become a hot topic in the study of brain cancer treatment. Among the various isothiocyanate compounds, it has been confirmed that benzyl isothiocyanate (BITC) can inhibit the growth of a variety of tumors, including leukemia, glioma and lung cancer, both inside and outside the body. This study explored inhibitory effects of BITC on human glioma U87MG cells, as well as potential mechanisms. It was found that BITC could inhibit proliferation, induce apoptosis and arrest cell cycling of U87MG cells. In addition, it inhibited the expression of SOD and GSH, and caused oxidative stress to tumor cells. Therefore, it is believed that BITC can inhibit the growth of U87MG cells outside the body. Its mechanism may be related to the fact that BITC can cause oxidative stress to tumor cells.

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

Apoptosis Induced by Benzyl Isothiocyanate in Gefitinib-Resistant Lung Cancer Cells is Associated with Akt/MAPK Pathways and Generation of Reactive Oxygen Species

Gefitinib is the first targeted drug approved for non-small cell lung cancer (NSCLC) treatment. Clinical trails showed that patients with certain clinical and histologic characteristics (such as women, patients of East Asian descent, no history of smoking, and adenocarcinoma) had higher rates of response and overall survival. Despite excellent clinical response to gefitinib in certain NSCLC patients, nearly all patients who respond initially to gefitinib later develop drug resistance. Isothiocyanates have been shown to possess antitumor activity, inhibiting several types of cancer cells growth. However, there are limited studies on their effects on chemoresistance of cancer cells. In this report, we found that benzyl isothiocyanate (BITC) inhibited gefitinib-resistant human NSCLC cells growth by inducing apoptosis in a dose-dependent manner, and activated caspase-3. There were no effects of BITC on epidermal growth factor receptor and multidrug resistant proteins expression. BITC caused cell cycle arrest at G2/M phase, reactive oxygen species generation, and glutathione depletion. Akt activity and NFκB transcriptional activation were suppressed; mitogen-activated protein kinase and activator protein 1 (AP-1) were activated. Our results demonstrated that BITC overcame gefitinib resistance in lung cancer cells. The further understanding of the anti-resistance mechanism of BITC would contribute to establish it as a potent lead compound for the synthesis of novel anticancer drugs.

Abstract 139: MicroRNA-375 and microRNA-221: Potential noncoding RNAs associated with antiproliferative activity of benzyl isothiocyanate in pancreatic cancer

Abstract Large high-throughput studies in patients with pancreatic cancer revealed that oncogenic miR-221 is commonly upregulated microRNA whereas putative tumor suppressor miR-375 is generally down modulated when compared to adjacent benign pancreatic tissue or normal pancreas. In our laboratory, we observed that deregulated expression of miR-375 and miR-221 (as noted in pancreatic cancer patients) can be recapitulated in PDX1-Cre+; KRasG12D+ pancreatic cancer mouse model. Coherent data obtained from pancreatic cancer patient samples to preneoplastic lesions developed in preclinical mouse model tempted us to postulate that aberrant expression of these miRNAs (−375 and-221) might play significant role for the development of pancreatic cancer. Interestingly, ablation of miR-221 by ectopic expression of antagomir-221 in pancreatic ductal carcinoma cells can significantly exert antiproliferative effect with increased expression of p27kip1, a target of miR-221. To the other end, premiR-375 overexpression diminished cell proliferation with downmodulation of Insulin like growth factor binding protein 5 (IGFBP5) and Caveolin-1 (CAV-1 gene), which are of particular interest because of their potential link with pancreatic cancer cell growth and diagnosis. It is quite intriguing that the levels of both CAV-1 and IGFBP5 expression were elevated when miR-375 expression wanes in KRASG12D driven PanIN lesion in compound mutant mice. Previous studies in our laboratory demonstrate that benzyl isothiocyanate (BITC), a prototypical member of the isothiocyanate (ITC) family, inhibits proliferation of human pancreatic cancer cells by causing cell cycle arrest and inducing apoptosis. Interestingly, real time qRT-PCR analysis indicates that anticancer agent BITC can diminish oncogenic miR-221 expression and elevate the level of tumor suppressor miR-375 in pancreatic cancer cells. Besides, genetic manipulation studies revealed that enforced silencing of miR-221 or overexpression of miR-375 enhances the antiproliferative effect of BITC in pancreatic ductal carcinoma cells. Next we explored the efficacy of BITC to interfere with the development of mouse PanIN lesions. Results indicate that pancreata of BITC administered compound mutant mice exhibit reduced level of IGFBP5 protein. Histopathological analysis of the extent of PanINs as well as studies on the magnitude of expression of miR-221/miR-375 and other molecular mediators in these pancreata are in progress. Explicitly, our investigation might provide new insight into nonconventional microRNA replacement therapy against pancreatic cancer. 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 139. doi:10.1158/1538-7445.AM2011-139

Effects of benzyl isothiocyanate on the reproduction of Meloidogyne incognita on Glycine max and Capsicum annuum

Abstract Reproduction of Meloidogyne incognita on either Capsicum annuum or Glycine max was suppressed when infective second-stage juveniles (J2) were exposed to 0.03 mM benzyl isothiocyanate (BITC) for 2 h prior to inoculation of the host. Infectivity as rated by a gall index was significantly reduced on both G. max and C. annuum. In C. annuum, nematode egg masses were recovered from 48% of the plants inoculated with BITC-treated J2 compared with 98% for the controls, and egg mass scores were reduced. Egg mass production was reduced in C. annuum by 69% and mean total eggs/plant was reduced by 97% in G. max. When plants were inoculated with treated J2, two measures of plant health, root weight and shoot weight, were reduced in C. annuum but not in G. max. However, root and shoot weights were not correlated with egg production for either host plant, irrespective of treatment. There were strong interactions between egg production, as measured by mean total eggs/plant (G. max) or mean total eggs/egg mass (C. annuum), and hatching of J2 from eggs obtained from all combinations of plant host and J2 treatment. Hatch of J2 from eggs obtained from G. max was significantly lower when plants had been inoculated with BITC-treated J2 than when plants had been inoculated with control J2. Such effects were not observed with C. annuum. BITC may have important residual consequences on the progeny of M. incognita not directly exposed to the chemical.

Effects of benzyl isothiocyanate and 2-phenethyl isothiocyanate on benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism in F-344 rats.

A mixture of dietary benzyl isothiocyanate (BITC) and 2-phenethyl isothiocyanate (PEITC) inhibits lung tumorigenesis by a mixture of benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Previous studies indicated that inhibition of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) releasing DNA adducts of NNK by PEITC in the lung was responsible for inhibition of tumorigenicity. We have now extended these investigations to F-344 rats treated with 2 p.p.m. B[a]P in the diet and 2 p.p.m. NNK in the drinking water. The effects of BITC (1 micromol/g diet), PEITC (3 micromol/g diet), and a mixture of BITC plus PEITC (1 and 3 micromol/g diet) on DNA and hemoglobin (Hb) adducts of B[a]P and NNK, and on two urinary metabolites of NNK, were examined. DNA adducts were quantified after 8 and 16 weeks of treatment. Hb adducts were quantified in blood samples withdrawn every 2 weeks. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide NNAL-Gluc were measured in urine every 4 weeks. PEITC or BITC plus PEITC significantly reduced levels of HPB releasing DNA adducts of NNK in lung at 8 and 16 weeks, but there was no effect of BITC. There were no effects of any of the treatments on levels of HPB releasing DNA adducts of NNK in liver, or on DNA adducts of B[a]P in either lung or liver. PEITC or BITC plus PEITC significantly inhibited the formation of Hb adducts of NNK from 2-12 weeks of treatment while there were no effects on Hb adducts of B[a]P. There was a significant increase in levels of NNAL and NNAL-Gluc in the urine of the rats treated with PEITC or BITC plus PEITC. These results demonstrate that dietary PEITC, or a mixture of BITC plus PEITC, inhibit the formation of HPB releasing adducts of NNK in the rodent lung, leading to inhibition of tumorigenesis.

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung.

Dietary phenethyl isothiocyanate (PEITC) and a mixture of dietary PEITC and benzyl isothiocyanate (BITC) inhibit lung tumorigenesis in A/J mice induced by a mixture of the tobacco smoke carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we tested the hypothesis that inhibition of tumorigenesis by these isothiocyanates was due to inhibition of DNA adduct formation. We quantified the following pulmonary DNA adducts: N2-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl]deoxyguanosine (BPDE-N2-dG) from B[a]P; and O(6)-methylguanine (O(6)-mG) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts from NNK. Initial experiments demonstrated that there were no effects of B[a]P on NNK-DNA adduct formation, or vice versa, and established by way of a time course study the appropriate sacrifice intervals for the main experiment. Dietary PEITC, or dietary BITC plus PEITC, inhibited the formation of HPB-releasing DNA adducts of NNK at several of the time points examined. There were no effects of dietary isothiocyanates on levels of O(6)-mG or BPDE-N2-dG. These results, which are consistent with previous studies in rats and with tumor inhibition data in mice, support a role for inhibition of HPB-releasing DNA adducts of NNK as a mechanism of inhibition of tumorigenesis by dietary PEITC and BITC plus PEITC. However, the observed inhibition was modest, suggesting that other effects of isothiocyanates are also involved in chemoprevention in this model.

Simultaneous Treatment With Benzyl Isothiocyanate, a Strong Bladder Promoter, Inhibits Rat Urinary Bladder Carcinogenesis by N-Butyl-N-(4-Hydroxybutyl)Nitrosamine

Effects of benzyl isothiocyanate (BITC) on urinary bladder carcinogenesis were examined in rats simultaneously treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 20 6-wk-old Fischer 344 male rats were given 10, 100, or 1,000 ppm BITC in the diet or a basal diet with 50 ppm BBN in the drinking water for 40 wk and then killed for autopsy. Additional groups consisting of 10 or 9 rats were similarly given BITC or the basal diet alone without BBN treatment. With BBN treatment, dysplasia, papilloma, and carcinoma incidences and multiplicities were dramatically decreased by simultaneous treatment with BITC in a clear dose-dependent manner. In contrast, epithelial hyperplasia was induced in rats treated with 100 and 1,000 ppm BITC without BBN. These results clearly indicate that although BITC may have weak carcinogenic potency, it is a potent chemopreventive agent against bladder tumor induction by BBN.

Modifying effects of benzyl isothiocyanate and benzyl thiocyanate on DNA synthesis in primary cultures of rat hepatocytes.

The effects of benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC) on two types of DNA synthesis were examined in hepatocyte primary cultures (HPC). Male F344 rats were fed BITC- or BTC-containing diets at a concentration of 400 p.p.m. Using hepatocytes isolated from these rats, DNA repair was measured by unscheduled DNA synthesis (UDS) for some genotoxic carcinogens, e.g. 2-acetylaminofluorene (AAF), methylazoxymethanol (MAM) acetate, 9,10-dimethyl-1,2-benzanthracene (DMBA) and diethylnitrosamine (DEN), and compared with that in the hepatocytes from rats without BITC or BTC treatment. Replicative DNA synthesis (RDS) was also evaluated in the hepatocytes of rats with or without thiocyanate treatment. Both BITC and BTC reduced UDS elicited by these carcinogens. The level of RDS in the hepatocytes of rats exposed to BITC or BTC was markedly lower than in the cells of rats without BITC or BTC exposure. These results indicate that in vivo exposure to BITC and BTC suppressed carcinogen-induced genotoxicity and cell proliferative activity and suggest that this assay may prove useful in detecting chemopreventive agents for cancer and in investigating the properties of carcinogenesis modifiers.