Cancer Preventive Isothiocyanates Induce Selective Degradation of Cellular ��- and ��-Tubulins by Proteasomes

Although it is conceivable that cancer preventive isothiocyanates (ITCs), a family of compounds in cruciferous vegetables, induce cell cycle arrest and apoptosis through a mechanism involving oxidative stress, our study shows that binding to cellular proteins correlates with their potencies of apoptosis induction. More recently, we showed that ITCs bind selectively to tubulins. The differential binding affinities toward tubulin among benzyl isothiocyanate, phenethyl isothiocyanate, and sulforaphane correlate well with their potencies of inducing tubulin conformation changes, microtubule depolymerization, and eventual cell cycle arrest and apoptosis in human lung cancer A549 cells. These results support that tubulin binding by ITCs is an early event for cell growth inhibition. Here we demonstrate that ITCs can selectively induce degradation of both α- and β-tubulins in a variety of human cancer cell lines in a dose- and time-dependent manner. The onset of degradation, a rapid and irreversible process, is initiated by tubulin aggregation, and the degradation is proteasome-dependent. Results indicate that the degradation is triggered by ITC binding to tubulin and is irrelevant to oxidative stress. This is the first report that tubulin, a stable and abundant cytoskeleton protein required for cell cycle progression, can be selectively degraded by a small molecule.Microtubules as a major cytoskeleton component in all eukaryotic cells play essential roles such as maintenance of cell polarity, intracellular traffic, organization, and cell motility (1–4). During cell division, the microtubule-formed mitotic spindle ensures the replicated chromosomes separate evenly at the end of the mitotic phase to the two daughter cells (1). It is because of its essential roles in cell growth that microtubules become a valid target for the development of anti-microtubule drugs against the rapidly growing cancer cells (2), as interference of microtubule dynamics arrests cell cycle progression and induces apoptosis (3). Therefore, microtubules have been considered one of the best targets to date for cancer chemotherapy (4).Isothiocyanates (ITCs)³ are among the best studied chemopreventive small molecules (5). The three most studied ITCs, including benzyl-ITC (BITC; abundant in garden cress), phenethyl-ITC (PEITC; in watercress), and sulforaphane (SFN; in broccoli sprouts), have been shown to induce apoptosis and cell cycle arrest (5–8). Although it is believed that oxidative stress plays a role in cell cycle arrest and apoptosis induced by ITCs (6–12), we found that binding to proteins is a predominant intracellular chemical reaction of ITCs, and their protein binding affinities correlate well with inhibition of cell proliferation and induction of apoptosis (13). Recently, we identified tubulin, the microtubule constituent, as an in vivo target of ITCs by two-dimensional gel electrophoresis and mass spectrometry (14). The growth inhibition of human non-small lung cancer A549 cells by ITCs followed the order of BITC > PEITC > SFN. The same order of potency was seen in their binding affinities toward tubulin, induction of its conformational changes, and inhibition of its polymerization. The study provides the first evidence of an in vivo ITC-tubulin binding adduct, indicating that direct modification of cysteines in tubulin by ITCs, rather than oxidative stress, may trigger cell cycle arrest and apoptosis.Here we report an unexpected novel finding that tubulin is selectively degraded in a variety of human cancer cells treated with ITCs. We provide evidence that the degradation is initiated by its binding with ITCs and mediated by the ubiquitin-proteasome pathway. Tubulin has long been viewed as a stable and abundant protein, and its levels in cells are tightly regulated (15). In the literature, the only studies on cellular tubulin level change are related to “the auto-regulation theory,” i.e. when microtubules collapse, the presence of a massive amount of tubulin monomers would selectively destabilize tubulin mRNA and subsequently decrease tubulin protein synthesis (16–18). To our knowledge, there is no report on tubulin degradation as a result of treatment with any agents. Our studies provide strong evidence that supports tubulin as a target of ITCs for cell growth inhibition, pointing to a new mechanism for the anti-microtubule or anti-mitosis effects of ITCs through covalent binding to tubulin and presenting a platform to study protein stability by modification with small molecules.     

Cell death induction by isothiocyanates and their underlying molecular mechanisms

An important and promising group of compounds that have a chemopreventive property are organosulfur compounds, such as isothiocyanates (ITCs). In recent years, it has been shown that ITCs induce apoptosis in various cancer cell lines and experimental rodents. During the course of apoptosis induction by ITC, multiple signal-transduction pathways and apoptosis intermediates are modulated. We have also clarified the molecular mechanism underlying the relationship between cell cycle arrest and apoptosis induced by benzyl isothiocyanate (BITC), a major ITC compound isolated from papaya. The exposure of cells to BITC resulted in the inhibition of the G2/M progression that coincided with not only the up-regulated expression of the G2/M cell cycle arrest-regulating genes but also the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2/M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC. We also found that BITC induced the cytotoxic effect more preferentially in the proliferating normal human colon epithelial cells than in the quiescent cells. Conversely, treatment with an excessive concentration of BITC resulted in necrotic cell death without DNA ladder formation. This review addresses the biological impact of cell death induction by BITC as well as other ITCs and the involved signal transduction pathways.     

Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells

In this study, we have developed a novel method to identify isothiocyanate (ITC)-targeted molecules using two well-studied ITCs: benzyl ITC (BITC) and phenethyl ITC (PEITC). The principle of this method is based on identifying a pattern of differences between BITC and PEITC given that they show similar chemical and biological behaviors. For method validation, dithiothreitol-reduced bovine insulin as a model molecule was incubated with either BITC or PEITC, and digested peptides were analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and liquid chromatography quadrupole TOF-MS (LC-Q-TOF-MS). Three peptides-NYCN, FVNQHLCGSHLVE, and ALYLVCGE-were identified as being adducted with BITC or PEITC on their cysteine residues. Each set of peptides adducted with either BITC or PEITC showed retention times (RT(BITC)相似文献   

Determination of new biomarkers to monitor the dietary consumption of isothiocyanates

Isothiocyanates (ITCs) found in cruciferous vegetables have been associated with a reduced cancer risk in humans. We determined serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethylisothiocyanate (PEITC) and sulforaphane (SFN) in 85 healthy men from a dietary, randomized, controlled trial. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using liquid chromatography–tandem mass spectrometry. At the beginning of the study (and after 4 weeks) 4.7% (2.4%), 48.2% (35.3%), 5.9% (10.6%), and 24.7% (23.5%) of the samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys and SFN-Lys, respectively. This method enables the quantification of ITC adducts in albumin from large, prospective studies on diet and cancer.     

Inhibition of Premature Death by Isothiocyanates through Immune Restoration in LP-BM5 Leukemia Retrovirus-Infected C57BL/6 Mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.     

Inhibition of premature death by isothiocyanates through immune restoration in LP-BM5 leukemia retrovirus-infected C57BL/6 mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.     

Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines

Organic isothiocyanates (ITCs) are dietary components present in cruciferous vegetables. The purpose of this investigation was to examine the cytotoxicity of 1-naphthyl isothiocyanate (NITC), benzyl isothiocyanate (BITC), beta-phenethyl isothiocyanate (PEITC), and sulforaphane in human breast cancer MCF-7 and human mammary epithelium MCF-12A cell lines, as well as in a second human epithelial cell line, human kidney HK-2 cells. The cytotoxicity of NITC, BITC, PEITC, and sulforaphane, as well as the cytotoxicity of the chemotherapeutic agents daunomycin (DNM) and vinblastine (VBL), were examined in MCF-7/sensitive (wt), MCF-7/Adr (which overexpresses P-glycoprotein), MCF-12A, and HK-2 cells. Cell growth was determined by a sulforhodamine B assay. The IC50 values for DNM and VBL in MCF-7/Adr cells were 7.12 +/- 0.42 microM and 0.106 +/- 0.004 microM (mean +/- SE) following a 48-hr exposure; IC50 values for BITC, PEITC, NITC, and sulforaphane were 5.95 +/- 0.10, 7.32 +/- 0.25, 77.9 +/- 8.03, and 13.7 +/- 0.82 microM, respectively, with similar values obtained in MCF-7/wt cells. Corresponding values for BITC, PEITC, NITC, and sulforaphane in MCF-12A cells were 8.07 +/- 0.29, 7.71 +/- 0.07, 33.6 +/- 1.69, and 40.5 +/- 1.25 microM, respectively. BITC and PEITC can inhibit the growth of human breast cancer cells as well as human mammary epithelium cells at concentrations similar to those of the chemotherapeutic drug DNM. Sulforaphane and NITC exhibited higher IC50 values. The effect of these ITCs on cell growth may contribute to the cancer chemopreventive properties of ITCs by suppressing the growth of preclinical tumors, and may indicate a potential use of these compounds as chemotherapeutic agents in cancer treatment.     

HDAC turnover,CtIP acetylation and dysregulated DNA damage signaling in colon cancer cells treated with sulforaphane and related dietary isothiocyanates

Histone deacetylases (HDACs) and acetyltransferases have important roles in the regulation of protein acetylation, chromatin dynamics and the DNA damage response. Here, we show in human colon cancer cells that dietary isothiocyanates (ITCs) inhibit HDAC activity and increase HDAC protein turnover with the potency proportional to alkyl chain length, i.e., AITC < sulforaphane (SFN) < 6-SFN < 9-SFN. Molecular docking studies provided insights into the interactions of ITC metabolites with HDAC3, implicating the allosteric site between HDAC3 and its co-repressor. ITCs induced DNA double-strand breaks and enhanced the phosphorylation of histone H2AX, ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase-2 (CHK2). Depending on the ITC and treatment conditions, phenotypic outcomes included cell growth arrest, autophagy and apoptosis. Coincident with the loss of HDAC3 and HDAC6, as well as SIRT6, ITCs enhanced the acetylation and subsequent degradation of critical repair proteins, such as CtIP, and this was recapitulated in HDAC knockdown experiments. Importantly, colon cancer cells were far more susceptible than non-cancer cells to ITC-induced DNA damage, which persisted in the former case but was scarcely detectable in non-cancer colonic epithelial cells under the same conditions. Future studies will address the mechanistic basis for dietary ITCs preferentially exploiting HDAC turnover mechanisms and faulty DNA repair pathways in colon cancer cells vs. normal cells.     

Benefits and Risks of the Hormetic Effects of Dietary Isothiocyanates on Cancer Prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1–5 µM) to promote cell proliferation to 120–143% of the controls in a number of human cell lines, whilst at high levels (10–40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10–20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint.     

Sulforaphane as a promising molecule for fighting cancer

A number of natural compounds with inhibitory effects on tumorigenesis have been identified from our diet. Several studies have documented the cancer-preventive activity of a significant number of isothiocyanates (ITCs), the majority of which occur in plants, especially in Cruciferous vegetables. The most characterized ITC is sulforaphane (SFN). SFN has received a great deal of attention because of its ability to simultaneously modulate multiple cellular targets involved in cancer development, including: (i) DNA protection by modulating carcinogen-metabolizing enzymes and blocking the action of mutagens; (ii) inhibition of cell proliferation and induction of apoptosis, thereby retarding or eliminating clonal expansion of initiated, transformed, and/or neoplastic cells; (iii) inhibition of neoangiogenesis, progression of benign tumors to malignant tumors, and metastasis formation. SFN is therefore able to prevent, delay, or reverse preneoplastic lesions, as well as to act on cancer cells as a therapeutic agent. Taking into account this evidence and its favorable toxicological profile, SFN can be viewed as a conceptually promising agent in cancer prevention and/or therapy.     

Administration of isothiocyanates enhances heat tolerance in Arabidopsis thaliana

Although it has been documented that plants generate isothiocyanates (ITCs) through the glucosinolate-myrosinase system to defend against biotic stresses, the roles of ITCs in defending against abiotic stresses have scarcely been studied. Here, we report that exogenously applied ITCs enhance the heat tolerance of Arabidopsis thaliana. Pre-administration of phenethyl ITC to Arabidopsis plants mitigated growth inhibition after heat stress at 55?°C for 1?h. Although methyl ITC and allyl ITC also tended to reduce the growth inhibition that the same heat treatment caused, the reduction effects were weaker. The expression levels of heat shock protein 70 genes in Arabidopsis were elevated after phenethyl ITC treatment. These results suggest that ITCs may act as heat-tolerance enhancers in plants.     

Proteomic identification of binding targets of isothiocyanates: A perspective on techniques

Intake of cruciferous vegetable is inversely associated with the risk of several cancer types. Isothiocyanates (ITCs) are believed to be important constituents contributing to these cancer-preventive effects. Although several mechanisms, including induction of apoptosis, have been proposed for the anti-carcinogenesis activities of ITCs, detailed upstream triggering events are still not fully understood. Identification of ITC binding targets in cellular proteins is crucial for not only mechanistic studies but also future drug screening and design. In this review, we summarize recent progress in discovery of ITC protein targets from a technical perspective. The advantages and limitations of each method are discussed to facilitate future studies on target discovery of ITCs and perhaps other compounds.     

High-performance liquid chromatography-based determination of total isothiocyanate levels in human plasma: application to studies with 2-phenethyl isothiocyanate

Dietary and pharmacologic isothiocyanates (ITCs) may play a role in reducing the risk of certain cancers. The quantification of ITCs in humans is important both for epidemiological and pharmacokinetic studies. We describe a modification of an HPLC-based assay of urinary ITCs for use with human plasma. The assay utilizes the cyclocondensation reaction of 1,2-benzenedithiol with ITCs present in human plasma, followed by a two-step hexane extraction and analysis by HPLC using UV detection at 365 nm. The method shows linearity and reproducibility with human plasma over a range of 49-3003 nM phenethyl isothiocyanate (PEITC) (r(2) = 0.996 +/- 0.003). A similar degree of linearity was seen with two other biologically occurring conjugates of PEITC: PEITC--N-acetylcysteine (PEITC--NAC) and PEITC--glutathione (PEITC--GSH). The recovery of PEITC assessed on multiple days was 96.6 +/- 1.5% and was 100% for PEITC--GSH and PEITC--NAC. The reproducibility of the assay on multiday samplings showed a mean %CV of 6.5 +/- 0.3% for PEITC, 6.4 +/- 4.3 for PEITC--NAC and 12.3 +/- 3.9 for PEITC--GSH. In clinical studies, mean plasma ITC level of 413 +/- 193 nM PEITC equivalents was determined for a non-dietary-controlled group of 23 subjects. Multiday analysis data from pharmacokinetic plasma sets of 3 subjects taking a single dose of PEITC at 40 mg showed a good CV (range: 16-21%). The applicability of the methodology to pharmacokinetic studies of PEITC in humans is demonstrated.     

Benzyl isothiocyanate modifies expression of the G2/M arrest-related genes

Naturally occurring isothiocyanates are effective chemoprotective agents against chemical carcinogenesis in experimental animals. In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis. The exposure of HL-60 cells to BITC resulted in the inhibition of the G2/M progression that coincided with the apoptosis induction. We demonstrated that BITC significantly up-regulated expression of the G2/M cell cycle arrest-regulating genes including p21, GADD45, and 14-3-3sigma. Thus, these gathered data further supported that BITC has a potential to induce apoptosis selectively in the proliferating pre-cancerous cells through a cell cycle arrest-dependent mechanism.     

Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting inhibitors

Histone deacetylases (HDACs) have proven to be promising targets for the development of anti-cancer drugs. In this study, we reported a series of novel chalcone based tubulin and HDAC dual-targeting inhibitors. Three compounds inhibited the activities of HDAC and tubulin polymerization simultaneously and displayed anti-proliferative activities toward eleven human tumor cell lines. Compound 8a remarkably induced growth inhibition, apoptosis and G2/M phase arrest of A549 tumor cells. Finally, the inhibitory activities of 8a against HDAC6 and tubulin were rationalized by molecular docking studies.     

Synthesis and biological evaluation of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides as antimitotic agents

A library of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides (7a–al) have been designed, synthesized and screened for their anti-proliferative activity against some selected human cancer cell lines namely DU-145, A-549, MCF-7 and HeLa. Most of them have shown promising cytotoxicity against lung cancer cell line (A549), amongst them 7f was found to be the most potent anti-proliferative congener. Furthermore, 7f exhibited comparable tubulin polymerization inhibition (IC₅₀ value 2.04 µM) to the standard E7010 (IC₅₀ value 2.15 µM). Moreover, flow cytometric analysis revealed that this compound induced apoptosis via cell cycle arrest at G₂/M phase in A549 cells. Induction of apoptosis was further observed by examining the mitochondrial membrane potential and was also confirmed by Hoechst staining as well as Annexin V-FITC assays. Furthermore, molecular docking studies indicated that compound 7f binds to the colchicine binding site of the β-tubulin. Thus, 7f exhibits anti-proliferative properties by inhibiting the tubulin polymerization through the binding at the colchicine active site and by induction of apoptosis.     

A cautionary note on using N-acetylcysteine as an antagonist to assess isothiocyanate-induced reactive oxygen species-mediated apoptosis

N-Acetylcysteine (NAC) has been widely used in cell culture-based studies for the role of reactive oxygen species (ROS) generation in apoptosis induction by isothiocyanates (ITCs). Here we have demonstrated, using [¹⁴C]phenethyl ITC and [¹⁴C]sulforaphane, that NAC pretreatment significantly reduces ITC cellular uptake by conjugating with ITCs in the medium, suggesting that reduced uptake of ITCs, rather than the antioxidant activity of NAC itself, is responsible for the diminished downstream apoptotic effects. The study provides a cautionary note on the assay in studying mechanisms of apoptosis by ITCs and other electrophilic and thiol-reactive compounds.