Candidate dietary phytochemicals modulate expression of phase II enzymes GSTP1 and NQO1 in human lung cells.
ABSTRACT: Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 micromol/L) for 24 h, 48 h, and 6 d, respectively. mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQO1 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQO1 mRNA expression in HBEC cells were similar. NQO1 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NQO1 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQO1 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing.
Project description:We show that naturally occurring isothiocyanates (ITCs) sensitize human non-small cell lung cancer cells to cisplatin. Moreover, the structure of the ITC side chain moiety is important for sensitization. In NCI-H596 cells, 20 microM benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) enhance the efficacy of various concentrations of cisplatin, but sulforaphane (SFN) does not. Reducing the concentration of BITC and PEITC to 10 microM still allows for the sensitization of cells to cisplatin. Neither cellular platinum accumulation nor DNA platination account for this increased cytotoxicity. BITC and PEITC deplete beta-tubulin, but SFN does not; this correlates with and may be important for sensitization.
Project description:Isothiocyanates, which are present as glucosinolate precursors in cruciferous vegetables, have strong activity against various cancers. Here, we compared the anti-metastatic effects of isothiocyanates (benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN)) by examining how they regulate MMP-9 expression. Isothiocyanates, particularly PEITC, suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 activity and invasion in various cancer cell lines. By contrast, N-methyl phenethylamine, a PEITC analog without an isothiocyanate functional group, had no effect. A reporter gene assay demonstrated that BITC, PEITC, and SFN suppressed TAP-induced MMP-9 expression by inhibiting AP-1 and NF-?B in U20S osteosarcoma cells. All three compounds reduced phosphorylation of FAK, ERK1/2, and Akt. In addition, MMP-9 expression was downregulated by inhibiting FAK, ERK1/2, and Akt. Isothiocyanates-mediated inhibition of FAK phosphorylation suppressed phosphorylation of ERK1/2 and Akt in U2OS and A549 cells, along with the translocation of p65 and c-Fos, suggesting that isothiocyanates inhibit MMP-9 expression and cell invasion by blocking phosphorylation of FAK. Furthermore, isothiocyanates, abolished MMP-9 expression and tumor metastasis in vivo with the following efficacy: PEITC>BITC>SFN. Thus, isothiocyanates act as anti-metastatic compounds that suppress MMP-9 activity/expression by inhibiting NF-?B and AP-1 via suppression of the FAK/ERK and FAK/Akt signaling pathways.
Project description:Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.
Project description:Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in a xenograft and transgenic mouse model, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigations but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 ?mol/L) resulted in downregulation of CXCR4 expression. None of the ITCs affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNAi of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma.
Project description:Glutathione reductase (GR), an essential antioxidant enzyme against oxidative stress, has become an attractive drug target for the development of anticancer and antimalarial drugs. In this regard, we evaluated the naturally occurring isothiocyanates as promising GR inhibitors and elucidated the mechanism of action. It was found that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) inhibited yeast GR (yGR) and human GR (hGR) in a time- and concentration-dependent manner. The <i>K<sub>i</sub></i> and <i>k<sub>inact</sub></i> of BITC against yGR were determined to be 259.87?µM and 0.0266?min<sup>-1</sup>, respectively. The GR inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis revealed that Cys<sup>61</sup> rather than Cys<sup>66</sup> at the active site of yGR was mono-benzyl thiocarbamoylated by BITC. Inhibition of intracellular GR by BITC and PEITC in cultured cancer cells was also observed. BITC and PEITC were evaluated as competitive and irreversible inhibitors of GR.
Project description:The reversible reaction of GSH with two dietary anticarcinogens, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), has been studied in the absence and presence of human glutathione S-transferases (GSTs). The spontaneous reaction at pH 7.4 and 37 degrees C yielded values for k2 of 17.9 and 6.0 M-1.s-1 for GSH conjugation of BITC and PEITC respectively (forward reaction), and k1 values of 6.9 x 10(-4) and 2.4 x 10(-4) s-1 for dissociation of the respective GSH conjugates, BITC-SG and PEITC-SG (reverse reaction). GSTs A1-1, A2-2, M1a-1a and P1-1 catalysed both the forward and reverse reactions with specific activities (mumol/min per mg at 30 microM isothiocyanate or GSH conjugate) ranging from 23.1 for the GSH conjugation of BITC by GST P1-1 to 0.03 for the dissociation of BITC-SG by GST A1-1. When present at similar concentration to substrates (12 microM), GSTs A1-1 and A2-2 but not GST M1a-1a shifted the equilibrium in favour of BITC-SG or PEITC-SG. Kinetic studies confirmed that GST A1-1 interacted selectively with the GSH conjugates in the micromolar range (Km 6.9 microM, Ki 4.3 microM), whereas GST M1a-1a interacted with BITC-SG and PEITC-SG with approx. 5-fold lower affinity. In conclusion, GSTs are true catalysts; at high intracellular concentration they also sequester GSH conjugates, promoting GSH conjugation, whereas trace extracellular GSTs promote dissociation of effluxed organic isothiocyanate-GSH conjugates.
Project description:BACKGROUND:The NAD(P)H:quinone oxidoreductase (NQO1) confers protection against semiquinones and also elicits oxidative stress. The C609T polymorphism of the NQO1 gene, designated NQO1*2, significantly reduces its enzymatic activity due to rapid degradation of protein. Since down regulation of NQO1 mRNA expression correlates with increased susceptibility for developing different types of cancers, we investigated the link between leukemia and the NQO1*2 genotype by mining a web-based microarray dataset, ONCOMINE. Phytochemicals prevent DNA damage through activation of phase II detoxification enzymes including NQO1. Whether NQO1 expression/activity in leukemia cells that carry the labile NQO1*2 genotype can be induced by broccoli-derived phytochemical sulforaphane (SFN) is currently unknown. METHODS AND RESULTS:The ONCOMINE query showed that: (1) acute lymphoblastic leukemia and chronic myelogenous leukemia are associated with reduced NQO1 levels, and (2) under-expressed NQO1 was found in human HL-60 leukemia cell line containing the heterozygous NQO1*2 polymorphism. We examined induction of NQO1 activity/expression by SFN in HL-60 cells. A dose-dependent increase in NQO1 level/activity is accompanied by upregulation of the transcription factor, Nrf2, following 1-10 ?M SFN treatment. Treatment with 25 µM SFN drastically reduced NQO1 levels, inhibited cell proliferation, caused sub-G1 cell arrest, and induced apoptosis, and a decrease in the levels of the transcription factor, nuclear factor-?B (NF?B). CONCLUSIONS:Up to 10 ?M of SFN increases NQO1 expression and suppresses HL-60 cell proliferation whereas ? 25 ?M of SFN induces apoptosis in HL-60 cells. Further, SFN treatment restores NQO1 activity/levels in HL-60 cells expressing the NQO1*2 genotype.
Project description:Naturally occurring isothiocyanates (ITCs) from cruciferous vegetables are widely studied for their cancer chemopreventive effects. In this study, we investigated the effects of ITCs on TLR signaling, and found that the two most promising ITCs, phenethyl ITCs (PEITC) and D,L-sulforaphane (SFN), have differential effects on dsRNA-mediated innate immune signaling through TLR3. PEITC preferentially inhibited TLR3-mediated IFN regulatory factor 3 (IRF3) signaling and downstream gene expression in vivo and in vitro, whereas SFN caused inhibition of TLR3-mediated NF-?B signaling and downstream gene expression. Mechanistically, PEITC inhibited ligand (dsRNA)-dependent dimerization of TLR3, resulting in inhibition of signaling through IFN regulatory factor 3. In contrast, SFN did not disrupt TLR3 dimerization, indicating that it affects further downstream pathway resulting in NF-?B inhibition. To examine the biological significance of these findings in the context of antitumor activities of these compounds, we used two approaches: first, we showed that dsRNA-mediated apoptosis of tumor cells via TLR3 was inhibited in the presence of PEITC, whereas this response was augmented by SFN treatment; second, in a separate assay measuring anchorage-independent growth and colony formation by immortalized fibroblasts, we made similar observations. Again in this study, PEITC antagonized dsRNA-mediated inhibition of colony formation, whereas SFN enhanced the inhibition. These results indicate biologically relevant functional differences between two structurally similar ITCs and may provide important insights in therapeutic development of these compounds targeted to specific cancer.
Project description:Isothiocyanates (ITCs), including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane, compounds found in cruciferous vegetable, are highly effective in inducing cell cycle arrest and apoptosis in a variety of cancer cells and animal models. Although some studies indicate that ITC-induced reactive oxygen species (ROS) generation may underlie apoptosis induction, our recent studies show that covalent binding to target proteins may be an important event triggering apoptosis. In this study, we report that BITC and PEITC significantly inhibit proteasome activity in a variety of cell types. Further studies show that ITCs inhibit both the 26S and 20S proteasomes, presumably through direct binding, and that this inhibition is unrelated to either ROS generation or ITC-induced protein aggregation. The potency of ITC-induced proteasome inhibition correlates with the rapid accumulation of p53 (tumor suppressor) and I?B nuclear factor-kappaB (nuclear factor-kappaB inhibitor). Finally, our results demonstrate that BITC and PEITC, the two strongest proteasome inhibitors, significantly suppress growth of multiple myeloma (MM) cells through induction of cell cycle arrest at G?/M phase and apoptosis. This study suggests that proteasome, like tubulin, is a potential molecular target of ITCs, thus providing a novel mechanism by which ITCs strongly inhibit growth of MM cells and new leads in identifying compounds with therapeutic and preventative efficacies for MM. It also supports the future studies of ITCs as therapeutic and preventive agents for MM.
Project description:Aromatic amines (AAs) are chemicals of industrial, pharmacological and environmental relevance. Certain AAs, such as 4-aminobiphenyl (4-ABP), are human carcinogens that require enzymatic metabolic activation to reactive chemicals to form genotoxic DNA adducts. Arylamine N-acetyltransferases (NAT) are xenobiotic metabolizing enzymes (XME) that play a major role in this carcinogenic bioactivation process. Isothiocyanates (ITCs), including benzyl-ITC (BITC) and phenethyl-ITC (PEITC), are phytochemicals known to have chemopreventive activity against several aromatic carcinogens. In particular, ITCs have been shown to modify the bioactivation and subsequent mutagenicity of carcinogenic AA chemicals such as 4-ABP. However, the molecular and biochemical mechanisms by which these phytochemicals may modulate AA carcinogens bioactivation and AA-DNA damage remains poorly understood. This manuscript provides evidence indicating that ITCs can decrease the metabolic activation of carcinogenic AAs via the irreversible inhibition of NAT enzymes and subsequent alteration of the acetylation of AAs. We demonstrate that BITC and PEITC react with NAT1 and inhibit readily its acetyltransferase activity (k(i) = 200 M(-1).s(-1) and 66 M(-1).s(-1) for BITC and PEITC, respectively). Chemical labeling, docking approaches and substrate protection assays indicated that inhibition of the acetylation of AAs by NAT1 was due to the chemical modification of the enzyme active site cysteine. Moreover, analyses of AAs acetylation and DNA adducts in cells showed that BITC was able to modulate the endogenous acetylation and bioactivation of 4-ABP. In conclusion, we show that direct inhibition of NAT enzymes may be an important mechanism by which ITCs exert their chemopreventive activity towards AA chemicals.