Dietary Intake Of Cruciferous Vegetables Research Articles

Current understanding and future directions of cruciferous vegetables and their phytochemicals to combat neurological diseases.

Neurological disorders incidences are increasing drastically due to complex pathophysiology, and the nonavailability of disease-modifying agents. Several attempts have been made to identify new potential chemicals to combat these neurological abnormalities. At present, complete abolishment of neurological diseases is not attainable except for symptomatic relief. However, dietary recommendations to help brain development or improvement have increased over the years. In recent times, cruciferous vegetables and their phytochemicals have been identified from preclinical and clinical investigations as potential neuroprotective agents. The present review highlights the beneficial effects and molecular mechanisms of phytochemicals such as indole-3-carbinol, diindolylmethane, sulforaphane, kaempferol, selenium, lutein, zeaxanthin, and vitamins of cruciferous vegetables against neurological diseases including Parkinson's disease, Alzheimer's disease, stroke, Huntington's disease, autism spectra disorders, anxiety, depression, and pain. Most of these cruciferous phytochemicals protect the brain by eliciting antioxidant, anti-inflammatory, and antiapoptotic properties. Regular dietary intake of cruciferous vegetables may benefit the prevention and treatment of neurological diseases. The present review suggests that there is a lacuna in identifying the clinical efficacy of these phytochemicals. Therefore, high-quality future studies should firmly establish the efficacy of the above-mentioned cruciferous phytochemicals in clinical settings.

Young Shoots and Mature Red Cabbage Inhibit Proliferation and Induce Apoptosis of Prostate Cancer Cell Lines

Prostate cancer is one of the most common cancers in men. Recent dietary and epidemiological studies have suggested the benefit of dietary intake of cruciferous vegetables in lowering the incidence of cancer. The health promoting effects of red cabbage are attributed to their mixture of phytochemicals known for their antioxidant and anticancer activity. In the current study, we investigated whether young shoots and mature red cabbage had any effect on prostate cancer cell lines (DU145 and LNCaP). Attempts were also made to identify the potential molecular mechanism(s) by which plant material elicits its biological effects on prostate cancer cell lines. Here we report that the studied vegetable inhibited the proliferation of cancer cells and that this process was associated with the induction of apoptosis via caspase-dependent and both extrinsic and intrinsic pathways. In addition, we also observed the regulation of genes and proteins associated with cell survival and apoptotic events.

Indoles Derived From Glucobrassicin: Cancer Chemoprevention by Indole-3-Carbinol and 3,3'-Diindolylmethane.

Hydrolysis of glucobrassicin by plant or bacterial myrosinase produces multiple indoles predominantly indole-3-carbinol (I3C). I3C and its major in vivo product, 3,3'-diindolylmethane (DIM), are effective cancer chemopreventive agents in pre-clinical models and show promise in clinical trials. The pharmacokinetics/pharmacodynamics of DIM have been studied in both rodents and humans and urinary DIM is a proposed biomarker of dietary intake of cruciferous vegetables. Recent clinical studies at Oregon State University show surprisingly robust metabolism of DIM in vivo with mono- and di-hydroxylation followed by conjugation with sulfate or glucuronic acid. DIM has multiple mechanisms of action, the most well-characterized is modulation of aryl hydrocarbon receptor (AHR) signaling. In rainbow trout dose-dependent cancer chemoprevention by dietary I3C is achieved when given prior to or concurrent with aflatoxin B1, polycyclic aromatic hydrocarbons, nitrosamines or direct acting carcinogens such as N-methyl-N'-nitro-nitrosoguanidine. Feeding pregnant mice I3C inhibits transplacental carcinogenesis. In humans much of the focus has been on chemoprevention of breast and prostate cancer. Alteration of cytochrome P450-dependent estrogen metabolism is hypothesized to be an important driver of DIM-dependent breast cancer prevention. The few studies done to date comparing glucobrassicin-rich crucifers such as Brussels sprouts with I3C/DIM supplements have shown the greater impact of the latter is due to dose. Daily ingestion of kg quantities of Brussels sprouts is required to produce in vivo levels of DIM achievable by supplementation. In clinical trials these supplement doses have elicited few if any adverse effects. Sulforaphane from glucoraphanin can act synergistically with glucobrassicin-derived DIM and this may lead to opportunities for combinatorial approaches (supplement and food-based) in the clinic.

Essential Role of Keap1-Nrf2 Signaling in Mood Disorders: Overview and Future Perspective.

Depression is one of the most common mood disorders with a high rate of relapse. Accumulating evidence suggests that the transcription factor Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1)-Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system plays a key role in inflammation which is involved in depression. Preclinical studies demonstrated that the protein expressions of Keap1 and Nrf2 in the prefrontal cortex (PFC), CA3 and dentate gyrus (DG) of hippocampus in mice with depression-like phenotype were lower than control mice. In the learned helplessness paradigm, the protein levels of Keap1 and Nrf2 in the PFC and DG of hippocampus from rats with depression-like phenotype were also lower than control and resilient rats. Furthermore, rodents with depression-like phenotype have higher levels of pro-inflammatory cytokines. Interestingly, Nrf2 knock-out (KO) mice exhibit depression-like phenotype, and higher serum levels of pro-inflammatory cytokines compared with wild-type mice. Furthermore, Nrf2 KO mice have lower expression of brain-derived neurotrophic factor (BDNF) in the PFC, and CA3 and DG of hippocampus compared to wild-type mice. 7,8-Dihydroxyflavone, a TrkB agonist, showed antidepressant effects in Nrf2 KO mice, by stimulating BDNF-TrkB in the PFC, CA3, and DG. Pretreatment with sulforaphane, a naturally occurring Nrf2 activator, prevented depression-like phenotype in mice after inflammation, or chronic social defeat stress. Interestingly, dietary intake of 0.1% glucoraphanin (a precursor of sulforaphane) containing food during juvenile and adolescent stages of mice could prevent depression-like phenotype in adulthood after chronic social defeat stress. Moreover, the protein expressions of Keap1 and Nrf2 in the parietal cortex from major depressive disorder and bipolar disorder were lower than controls. These findings suggest that Keap1-Nrf2 system plays a key role in the stress resilience which is involved in the pathophysiology of mood disorders. It is, therefore, possible that dietary intake of cruciferous vegetables including glucoraphanin (or SFN) may prevent or minimize relapse from remission, induced by stress and/or inflammation in depressed patients. In the review, the author would like to discuss the role of Keap1-Nrf2 system in mood disorders.

Sulforaphane Modulates AQP8-Linked Redox Signalling in Leukemia Cells.

Sulforaphane, a biologically active isothiocyanate compound extracted from cruciferous vegetables, has been shown to exert cytotoxic effects on many human cancer cells, including leukemia. However, the exact molecular mechanisms behind the action of sulforaphane in hematological malignancies are still unclear. Like other cancer cells, leukemia cells produce high level of reactive oxygen species; in particular, hydrogen peroxide derived from Nox family is involved in various redox signal transduction pathways, promoting cell proliferation and survival. Recent evidence show that many tumour cell types express elevated level of aquaporin isoforms, and we previously demonstrated that aquaporin-8 acts as H2O2 transport facilitator across the plasma membrane of B1647 cells, a model of acute myeloid human leukemia. Thus, the control of AQP8-mediated H2O2 transport could be a novel strategy to regulate cell signalling and survival. To this purpose, we evaluated whether sulforaphane could somehow affect aquaporin-8-mediated H2O2 transport and/or Nox-mediated H2O2 production in B1647 cell line. Results indicated that sulforaphane inhibited both aquaporin-8 and Nox2 expression, thus decreasing B1647 cells viability. Moreover, the data obtained by coimmunoprecipitation technique demonstrated that these two proteins are linked to each other; thus, sulforaphane has an important role in modulating the downstream events triggered by the axis Nox2-aquaporin-8. Cell treatment with sulforaphane also reduced the expression of peroxiredoxin-1, which is increased in almost all acute myeloid leukemia subtypes. Interestingly, sulforaphane concentrations able to trigger these effects are achievable by dietary intake of cruciferous vegetables, confirming the importance of the beneficial effect of a diet rich in bioactive compounds.

Abstract 2622: A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies

Abstract Epidemiological studies have demonstrated an inverse relationship between the intake of cruciferous vegetables and risk of cancers. The protective effects may be attributed to glucosinolates and glucobrassicins in these vegetables. While glucosinolates and glucobrassicins are not chemopreventive, their metabolites, isothiocyanates (ITCs), indole-3-carbinol (I3C), and 3,3′-diindolylmethane (DIM), have been shown to inhibit tumorigenesis in rodent models. In the past studies the total ITCs intake was estimated through food frequency questionnaires and/or urinary total ITC by a HPLC-based method. Self-reported intake of cruciferous vegetables has inherent limitations in estimating the intake of ITCs because of differences in the contents of their precursors present in different kinds of cruciferous vegetables, different locations and methods of cultivation, or different methods of food preparation. Animal and cell culture studies have shown that different types of ITCs have different anticancer properties and potency, suggesting that they are not equal in protecting against the development of cancers. The lack of a reliable method to quantify specific ITCs derived from dietary intake of cruciferous vegetables hinders the investigation of the role of specific ITCs against the development of cancer in human populations. To address this deficiency, we developed a flexible, highly sensitive, fast and reliable quantitative liquid chromatography mass spectrometry method (LC-MS/MS) for detecting and quantifying common dietary ITCs plus DIM in human urine. Urinary ITCs are measured as mercapturic acid conjugates (ITCs-NAC), predominant metabolite of ITCs, using multiple reaction monitoring (MRM) technique and deuterated [2H3]-ITCs-NAC as internal standards. DIM is quantified in separate experiment using [2H2]-DIM internal standard. The method was validated by a small set of previously analyzed samples from Singapore Chinese Health Study. We found an excellent correlation between previously reported total ITCs levels and sum of ITCs detected by newly developed method. The combination of minimal sample preparation, ultra-performance liquid chromatography, and highly sensitive mass spectrometer resulted in a method that is fast (below 10 min for ITCs and 12 min for DIM) and sensitive (low femtomolar LOQ) amenable to population studies. Citation Format: Marcin Dyba, Jian-Min Yuan, Jennifer Adams-Haduch, Fung-Lung Chung. A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies. [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 2622.

P594 PROMOTION OF LIVER INJURY AND FIBROSIS BY THE LIVER-DERIVED PLASMA PROTEIN HISTIDINE-RICH GLYCOPROTEIN IS BASED ON POLARIZING HEPATIC MACROPHAGES TOWARDS THE INFLAMMATORY M1 PHENOTYPE

cytokines secretion. In vitro study, DIM inhibited ConA-induced proliferation of splenic CD4+T cells, DIM enhanced the protein expression of CYP1A1 and CYP1B1, and decreased which of TLR4 on Con A-stimulated splenic CD4+T cells. Furthermore, blocking AhR with CH223191 significantly reversed the ability of DIM to promote the differentiation of Tregs, and caused marked inhibition of Th17 differentiation. On the other hand, blockage with single anti-TLR4 neutralizing antibody show the similar immune-regulatory effects of DIM to induc the production of Tregs. Conclusions: DIM may be an efficient theraprutic candidate of liver fibrosis, and high dietary intake of cruciferous vegetables may be an effective nutritional supplement for the patients with chronic liver disease.

Abstract B07: A meta-analysis of GSTM1 human genome as risk factor in lung cancer prevention

Abstract Purpose: Lung cancer is the highest cause of cancer related death in the United States. Its development is attributed to environmental and genetic factors. Smoking, diet, and occupation are environmental factors that can affect its development. Besides the environmental elements, the genetic aspect of this risk has also been identified as a major factor. Polymorphism in an individual's genotype has been recognized as a possible risk for lung cancer. GSTM1 has been one of the genes being studied with regards to genetic polymorphism affecting lung cancer development. A meta-analysis was conducted to examine the association of GSTM1 human genome as a risk factor for lung cancer. Procedure: Literature searches were completed by searching at three different times using keyword related to human GSTM1 and lung cancer. The searches generated 326 papers. The preliminary count includes 138 case-control studies. Quality rating of the literatures included will be based on Quality of Reporting of Meta-analysis (QUOROM). GSTM1 genes are categorized into present and null deletion genotype. Each category will be grouped by country and ethnicity. Inter-rater evaluation will be done for quality rating and gene count to ensure accurate appraisal of data collected. Pooled relative risks will be computed for each genotype and ethnicity to determine association with lung cancer. Findings: With the total cases of 12,288 and control subjects of 16,836, this meta-analysis is able to ascertain the association of GSTM1 with lung cancer risk. The present genotype of GSTM1 has been established as protective in nature (P < 0.05). In addition, the relative risk for the null genotype showed significance (P < 0.05) affirming its association to increased risk of developing lung cancer. In examining the subgroups for the study, only the Asian group shows a significant relative risk for both present and null genotype. Additionally, among the three Asian countries in the group, only China showed a significant relative risk for lung cancer development in the null genotype and it did not show significant relative risk for the protective genotype. On the contrary, Japan showed significance in association with the protective genotype. Further reviews confirmed that dietary factor such as intake of cruciferous vegetables, fruits, and green teas affect the development of lung cancer. Conclusion: The association between GSTM1 and lung cancer has been established through this meta-analysis. The results from this meta-analysis prompt the need for further studies that examine GSTM1 genotypes with inclusion of more studies for various race-ethnicity groups and countries. GSTM1 present genotype has an essential role in preventing lung cancer. There is a need to identify ways to enhance or maintain this GSTM1 genotype in future studies. Dietary intake of cruciferous vegetables is a promising factor in preventing lung cancer. Citation Format: Maria Suarez, S. Pamela Shiao, Mildred Gonzales, Amanda Lie, Ching-Yi Chiu. A meta-analysis of GSTM1 human genome as risk factor in lung cancer prevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B07.

Benzyl Isothiocyanate: Double Trouble for Breast Cancer Cells

An inverse association between dietary intake of cruciferous vegetables and cancer risk has been established for different types of malignancies, including breast cancer. The anticarcinogenic effect of cruciferous vegetables has been attributed to chemicals with an isothiocyanate (ITC) functional moiety. Research over the past three decades has provided extensive preclinical evidence for the efficacy of various ITCs against cancer in preclinical models. Benzyl isothiocyanate (BITC) is one such compound with the ability to inhibit chemically induced cancer, oncogenic-driven tumor formation, and human tumor xenografts in rodent cancer models. Prior work also has established that BITC has the ability to influence carcinogen metabolism and signaling pathways relevant to tumor progression and invasion. In this issue, Kim and colleagues show that BITC inhibits breast cancer stem cell growth, both in vitro and in vivo, in association with suppression of the full-length receptor tyrosine kinase RON as well as its activated truncated form (sfRon), both of which seem to drive stemness in breast cancer cells. Overexpression of RON or sfRon prevented the BITC effect. These data complement prior work from this group showing elimination of mammary tumor cells via tumor cell apoptosis by BITC administration. The inhibition of breast cancer stem cells is observed at pharmacologic concentrations of BITC. This perspective briefly reviews epidemiologic evidence, preclinical efficacy data, and molecular and cellular mechanistic attributes of BITC. Critical issues relevant to clinical development of BITC are discussed briefly.

Abstract A02: Meta-analyses of MPO, EGFR, and GSTM1 genes as risk factors for lung cancer prevention

Abstract Purpose: The purpose of this meta-analysis was to identify genes that have been associated with lung cancer (LC), and the risk factors associated with these genes for cancer prevention. LC is the leading cause of cancer related deaths worldwide. Three genes identified through the genome application framework, and well published in the literature, are Myeloperoxidase (MPO), a receptor for members of the epidermal growth factor (EGFR) family, and a glutathione S-transferase that belongs to the mu class (GSTM1). Findings from previous meta-analyses on this topic published from 10 years ago to 2 years ago were mixed with inconsistent and controversial findings. Since then, additional literature showed identification of the potential risk factors for the application of cancer genome and LC prevention. Procedure: On-line literature were searched from PubMed (PM), PM Central, PM Health databases for systematic reviews and meta-analyses by using a variety of combinations of key words about LC genes, cancer prevention, human models, and clinical trials. For this analysis, 61 studies were identified that have been published within the last 15 years and included data containing both control and LC cases of human samples. These papers were reviewed and evaluated using the criteria for the quality of studies. These studies included 20, 22, and 19 studies on MPO, EGFR and GSTM1 respectively. Summary of Data Findings: Preliminary analyses included 83,711 cases and 16,256 controls associating these genes with LC; with 6,363, 7,192, and 70,156 LC cases versus 7,3,46, 7,532, and 1,378 controls for MPO, EGFR and GSTM1 respectively. These studies included samples of all races and many ethnicity groups, and both genders. For prevention, these three genes each have unique metabolic mechanisms and major single nucleotide polymorphisms (SNPs) associated with the prognosis and treatment effects of LC. MPO is released from neutrophils in response to various pulmonary insults such as tobacco smoking and chemicals that produce air populations, thus, smokers and industrial workers have worse prognosis. Specifically, the MPO SNP, G-463A (rs2333227) has been significantly associated with LC risk. EGFR protein production including activating somatic mutations (exon 19 deletion and exon 21 L858R) may be different across gender and racial ethnic groups and demonstrate different sensitivity in non-smokers. Thus, prevention and treatments targeting EGFR may be considered in preference to other cancer treatments. Those with LC and SNPs of GSTM1 type have been found to be highly influenced by the dietary intake of cruciferous vegetables or fruits and green teas for the prognosis, especially for individuals who are susceptible for oxidative stress and oxidative DNA damage. Conclusion: Following analysis of these studies, it can be recommended that primary prevention of LC could include having a diet rich in antioxidants, and avoiding tobacco smoking and air pollution in environmental exposures. Meta-analysis of these studies has important impact for healthy lifestyles, health habits and screening. Maintenance of healthy lifestyles includes avoiding tobacco smoking or second hand smoking, and inhaling air pollutants; and taking a nutritional diet rich in antioxidants such as cruciferous vegetables or fruits and green teas. These preventive application measures are important in attenuating oxidative stress and DNA damage to reduce the risk factors of LC for the general public. Citation Format: Nick DeVries, S. Pamela Shiao, Melissa J. LaBonte, Ching-Yi Chiu, Linda H. Chiang, Rose Sakamoto. Meta-analyses of MPO, EGFR, and GSTM1 genes as risk factors for lung cancer prevention. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A02.

Cancer chemoprevention with dietary isothiocyanates mature for clinical translational research

Inverse association between dietary intake of cruciferous vegetables and cancer risk observed in population-based case-control studies is partly attributable to structurally simple but mechanistically complex phytochemicals with an isothiocyanate (-N=C=S) functional group. Cancer protective role for dietary isothiocyanates (ITCs) is substantiated by preclinical studies in rodent models. A common feature of many naturally occurring ITCs relates to their ability to cause growth arrest and cell death selectively in cancer cells. At the same time, evidence continues to accumulate to suggest that even subtle change in chemical structure of the ITCs can have a profound effect on their activity and mechanism of action. Existing mechanistic paradigm stipulates that ITCs may not only prevent cancer initiation by altering carcinogen metabolism but also inhibit post-initiation cancer development by suppressing many processes relevant to tumor progression, including cellular proliferation, neoangiogenesis, epithelial-mesenchymal transition, and self-renewal of cancer stem cells. Moreover, the ITCs are known to suppress diverse oncogenic signaling pathways often hyperactive in human cancers (e.g. nuclear factor-κB, hormone receptors, signal transducer and activator of transcription 3) to elicit cancer chemopreventive response. However, more recent studies highlight potential adverse effect of Notch activation by ITCs on their ability to inhibit migration of cancer cells. Mechanisms underlying ITC-mediated modulation of carcinogen metabolism, growth arrest, and cell death have been reviewed extensively. This article provides a perspective on bench-cage-bedside evidence supporting cancer chemopreventive role for some of the most promising ITCs. Structure-activity relationship and mechanistic complexity in the context of cancer chemoprevention with ITCs is also highlighted.

6-n-Propylthiouracil taster status not related to reported cruciferous vegetable intake among ethnically diverse children

Sensitivity to the taste of 6-n-propylthiouracil (PROP) (a bitter chemical related to the phenylthiocarbamide found in cruciferous vegetables) has been related to dietary intake or preferences of cruciferous vegetables among adults and young children but not middle-aged children or adolescents. We hypothesized that PROP taste sensitivity is related to lower reported dietary intake of cruciferous vegetables, primarily among younger children (ie, a moderating effect of child age). This study examined the relationship of PROP sensitivity to reported dietary intake across 3 days in 2 age groups of youth (9-10 and 17-18 years) while statistically controlling for physical activity, social desirability, and reporting bias. Cross-sectional design was used with a multiethnic (white, African American, Hispanic, etc) sample of 843 men and women. Children were recruited from and data were collected in local elementary and high schools that had at least 30% ethnic minority enrollment. Children providing nonplausible reports of dietary intake were deleted from the analyses. Body mass index was calculated and expressed in z scores. Energy intake and physical activity were measured by 3 telephone-conducted 24-hour dietary recalls with the Nutrient Data System for Research and 5 days of Actigraph (ActiGraph, Shalimar, Florida) activity monitor. The primary analyses included 347 students. 6-n-Propylthiouracil sensitivity was not related to intake of cruciferous vegetables. Intakes of the cruciferous vegetables were low, which may explain the lack of relationship.

Analysis of the isothiocyanates present in three Chinese Brassica vegetable seeds and their potential anticancer bioactivities

Epidemiological studies give evidence that high dietary intake of cruciferous vegetables has been associated with lower risk of cancer, this activity due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isothiocyanates (ITCs). In this study, the isothiocyanates contents and the anticancer activity of ethyl acetate extracts from three kinds of Chinese Brassica vegetable seeds have been examined and analyzed. A correlation was found between the potency of anticancer activity and the amount of isothiocyanates in their extracts. Extracts followed by GC–MS analysis revealed that 3-BITC (3-butenyl isothiocyanate) was mainly found in Chinese kale contained 77.58 ± 7.28 mg/g (dry weight); AITC (allyl isothiocyanate), 3-BITC and iberverin were the predominant isothiocyanates in Oxheart cabbage contained 33.57 ± 3.94, 26.50 ± 1.92 and 22.77 ± 0.00 mg/g, respectively; 3-BITC and sulforaphane were found as the major isothiocyanates in broccoli contained 138.52 ± 3.42 and 49.77 ± 0.18 mg/g, respectively. The amount of total isothiocyanates in broccoli was twice higher than that in other two species. The anticancer activity of broccoli was the highest, and the IC50 value of the extract inhibiting on the growth of A549, LAC, HELA and HepG2 were 14.38 ± 0.35, 10.38 ± 0.34, 19.45 ± 1.72 and 26.75 ± 0.82 mg/g, respectively. The extracts of Chinese Brassica vegetable seeds have the potential in inducing cancer cells apoptosis by morphology observation. The results of this study got a new kind of Brassica vegetable seeds that could produce high content of isothiocyanates, which is important to preparation of anticancer food additives.

Abstract B74: Phenethyl isothiocyanate and 4-phenylbutyl isothiocyanate inhibit the proliferation and viability of human renal carcinoma cells in vitro

Abstract Epidemiological studies have demonstrated a protective association between dietary intake of cruciferous vegetables and the incidence of renal cell carcinoma. Isothiocyanates (ITCs) are a class of sulfur-containing phytochemicals found uniquely and abundantly in cruciferous vegetables (broccoli, cauliflower, brussel sprouts, etc). We hypothesize that the inhibition of renal carcinoma cell growth and viability by ITCs underlies the protective effect of cruciferous vegetables on renal cell carcinoma. To test this hypothesis, we examined the effects of two ITCs: the naturally-occurring phenethyl ITC (PEITC) and the synthetic 4-phenylbutyl ITC (PBITC) on the Caki-1 human renal carcinoma cell line. Both PEITC and PBITC inhibited the proliferation of Caki-1 cells in a concentration-dependent manner. Cell cycle analysis revealed that PEITC and PBITC caused an arrest in the G2/M phase. Additionally, higher concentrations of PEITC and PBITC were cytotoxic to Caki-1 cells (IC50 of 7.0 and 4.8 µM, respectively). PEITC and PBITC induced caspase-3/7 activity in a concentration- and time-dependent manner, indicating that the ITCs induce apoptosis in the cells. These results indicate that PEITC and PBITC may be efficacious against renal cell carcinoma and suggest that the preventive effect of diets rich in cruciferous vegetables is due to their ITC content. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B74.

Benzyl isothiocyanate inhibits murine WEHI-3 leukemia cells in vitro and promotes phagocytosis in BALB/c mice in vivo

Many evidences have shown that dietary intake of cruciferous vegetables could protect against the risk of various types of malignancies. Benzyl isothiocyanate (BITC), one of the compounds from cruciferous vegetables, had shown induced cell cycle arrest and apoptosis in cancer cells. However, there is no available information to address that BITC affects murine leukemia cells in vitro and in vivo. Here, we investigated in vitro effects of BITC on murine leukemia WEHI-3 cells. BITC decreased the percentage of viable cells via G0/G1 arrest and apoptosis in WEHI-3 cells. BITC induced apoptosis through the dysfunction of mitochondria (decreased the levels of mitochondria membrane potential) and activation of caspase-3. Then we investigated in vivo effects of BITC on murine leukemia WEHI-3 cells and the results indicated that BITC decreased the weights of liver and spleen and it also decreased the percentage of CD11b and Mac-3 markers, indicating that the differentiation of the precursor of macrophage and B cells was inhibited. BITC promoted the activity of macrophage phagocytosis in cells which are isolated from PBMC and peritoneal (i.p.). Taken together, BITC can affect WEHI-3 cells in vitro and in vivo.

Benzyl Isothiocyanate Targets Mitochondrial Respiratory Chain to Trigger Reactive Oxygen Species-dependent Apoptosis in Human Breast Cancer Cells

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 (JNKK2(AA)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.

Methyl-3-indolylacetate inhibits cancer cell invasion by targeting the MEK1/2-ERK1/2 signaling pathway

Epidemiologic studies have suggested an inverse correlation between dietary intake of cruciferous vegetables and cancer risk. It is thus of interest to investigate the anticancer potential of phytochemicals presented in cruciferous vegetables. In this study, methyl-3-indolylacetate (MIA), a cruciferous indole for which the bioactivity has not been previously reported, was found to significantly suppress the invasion of cancer cells stimulated by the 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Our data show that MIA pretreatments inhibited matrix metalloproteinase 9 (MMP-9) expression in a concentration-dependent manner, resulting in decreased MMP-9 activity. By using real-time reverse transcription-PCR, luciferase reporter gene assay, and electrophoretic mobility shift assay, we provided convincing evidence that MIA suppresses MMP-9 gene transcription via targeting the activator protein-1 signaling but not the nuclear factor-kappaB pathway. The TPA-induced mitogen-activated protein kinase (MAPK) activation cascade was also analyzed. Despite extensive activation of major MAPKs [c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase-1/2 (ERK1/2)] under TPA stimulation, only the ERK1/2 activation and its consequent nuclear translocation were found to be diminished by MIA. Interestingly, MIA did not affect the TPA-induced phosphorylation of either c-Raf or MAPK/ERK kinase-1/2 (MEK1/2), two upstream kinases of ERK. Moreover, using the in vitro kinase assay, MIA was shown to inhibit the kinase activity of MEK1/2, the upstream kinases of ERK, suggesting that MEK is the major molecular target of MIA. In conclusion, data from this study provided new insight into the anticancer potential of MIA, a cruciferous vegetable-derived indole compound.

Benzyl isothiocyanate–induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak

Epidemiologic studies have revealed an inverse correlation between dietary intake of cruciferous vegetables and the risk of breast cancer. We now show that cruciferous vegetable constituent benzyl isothiocyanate (BITC) effectively suppresses growth of cultured human breast cancer cells (MDA-MB-231 and MCF-7) by causing G(2)-M phase cell cycle arrest and apoptosis induction. On the other hand, a normal mammary epithelial cell line (MCF-10A) is significantly more resistant to growth arrest and apoptosis by BITC compared with breast cancer cells. The BITC-mediated cell cycle arrest was associated with a decrease in levels of proteins involved in regulation of G(2)-M transition, including cyclin B1, cyclin-dependent kinase 1, and cell division cycle 25C. The BITC-induced apoptosis correlated with induction of proapoptotic proteins Bax (MCF-7) and Bak (MDA-MB-231 and MCF-7) and down-regulation of antiapoptotic proteins Bcl-2 and Bcl-xL (MDA-MB-231). The SV40-immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to BITC-induced DNA fragmentation compared with wild-type mouse embryonic fibroblasts. The BITC treatment caused rapid disruption of the mitochondrial membrane potential, leading to cytosolic release of apoptogenic molecules, which was accompanied by formation of autophagosome-like structures as revealed by transmission electron microscopy. The BITC-mediated apoptosis was associated with generation of reactive oxygen species and cleavage of caspase-9, caspase-8, and caspase-3. Apoptosis induction by BITC was significantly attenuated in the presence of a combined superoxide dismutase and catalase mimetic EUK134 as well as caspase inhibitors. In conclusion, the present study reveals a complex signaling leading to growth arrest and apoptosis induction by BITC.

Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells

A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer. In the current study, we examined the inhibitory effects of broccoli ( Brassica oleracea var. italica) and watercress ( Rorripa nasturtium aquaticum) extracts on 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed TPA-induced MMP-9 activity and invasiveness in a concentration dependant manner as determined by zymographic analysis. Furthermore, fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9 activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables.

Sulforaphane-induced Cell Death in Human Prostate Cancer Cells Is Initiated by Reactive Oxygen Species

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.