Proteomic analysis of covalent modifications of tubulins by isothiocyanates.

Although isothiocyanates (ITC), which are found in cruciferous vegetables, have been shown to inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in tumor cells, the biochemical mechanisms of cell growth inhibition by these compounds are not fully understood. Studies have reported that ITC binding to intracellular proteins may be an important event for initiating apoptosis. Specific protein target(s) and molecular mechanisms for ITC have been investigated in human lung cancer A549 cells using proteomic tools. Cells were treated with various amounts (1-100 µmol/L) of radiolabeled phenethyl-ITC (PEITC) and sulforaphane (SFN) and the extracted proteins resolved using 2-dimensional gel electrophoresis. The results of mass spectrometric analyses suggested that tubulin may be an in vivo binding target for ITC. The binding of ITC to tubulin was associated with growth arrest. The proliferation of A549 cells was significantly reduced by ITC, with benzyl-ITC (BITC) having a greater relative activity than PEITC or SFN. Mitotic arrest and apoptosis as well as disruption of microtubule polymerization were induced in the order: BITC > PEITC > SFN. An analysis of tubulins isolated from BITC-treated A549 cells showed that Cys(347), a conserved cysteine in all α-tubulin isoforms, was covalently modified by BITC. Taken together, these results suggest that tubulin is a binding target of ITC and that this interaction can lead to growth inhibition and apoptosis.

Discussion on the possibility of multi-layer intelligent technologies to achieve the best recover of musculoskeletal injuries: Smart materials, variable structures, and intelligent therapeutic planning.

Although intelligent technologies has facilitated the development of precise orthopaedic, simple internal fixation, ligament reconstruction or arthroplasty can only relieve pain of patients in short-term. To achieve the best recover of musculoskeletal injuries, three bottlenecks must be broken through, which includes scientific path planning, bioactive implants and personalized surgical channels building. As scientific surgical path can be planned and built by through AI technology, 4D printing technology can make more bioactive implants be manufactured, and variable structures can establish personalized channels precisely, it is possible to achieve satisfied and effective musculoskeletal injury recovery with the progress of multi-layer intelligent technologies (MLIT).

Cognitive therapeutic exercise in early proprioception recovery after knee osteoarthritis surgery.

Objective: This research aims to explore the therapeutic effect of cognitive therapeutic exercise (CTE) in proprioception recovery after knee osteoarthritis (KOA) surgery. Methods: In total, thirty-seven patients recovering from KOA surgery (including 27 patients who had undergone high-tibial osteotomy (HTO) procedure and 10 patients who had received total knee arthroplasty (TKA) treatment were randomly assigned to two groups: 18 patients in the CTE group and 19 patients for the control group (non-CTE). Patients in the CTE group received proprioceptive training as cognitive therapy to facilitate proprioception recovery for up to 4 weeks: 5 days a week and two 10-min sessions a day. Except for cognitive therapeutic exercise, the NCTE group and CTE group had the same treatment protocols. All the interventions began with permission from the surgeon-in-charge. In this research, we applied the joint repositioning training or joint-matching tasks, which is part of the proprioceptive training as a measurement for a proprioceptive training result where patients moved their knee joint from 0° (completely straight knee joint) to produce a presented joint angle, such as 30, 60, and 90° of flexion. Joint-matching task results were recorded before the treatment, at 2 and 4 weeks, postoperatively. The absolute difference between the results of these exercises and the knee flexion angle targets will be measured at each test-pre-rehabilitation (Pre-Reha), 2 weeks post-rehabilitation (2 weeks post-Reha), and 4 weeks post-rehabilitation (4 weeks post-Reha). Results: The absolute difference in the CTE group was significantly smaller than that of the control group after 4 weeks of treatment (P < 0.05). After 2 weeks of cognitive therapeutic exercise, the absolute difference between patients' exercises of joint repositioning and the target angle of 30° in the CTE group was smaller than that of the NCTE group (P < 0.01). After 4 weeks of therapy, the joint position sense (JPS) among patients who received cognitive therapeutic exercise when performing joint repositioning at angles of 30 and 60° were better improved than those without receiving proprioceptive training with the absolute difference smaller than those of the control group (P < 0.05). Conclusion: The joint reposition training provided for the CTE group is a painless proprioceptive training practice. This method is simple and effective, making it easy for patients to understand the purpose of training and improve patient engagement. The research showed that after 4 weeks of rehabilitation and physical training, the proprioception sense of both the NCTE and CTE groups improved significantly, and the efficacy of proprioceptive training in the CTE group was better than that of the NCTE group, which provided a new approach to the early proprioception recovery of a patient with KOA after surgery.

Isothiocyanates inhibit proteasome activity and proliferation of multiple myeloma cells.

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 IκB 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 G2/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.

Proteins as binding targets of isothiocyanates in cancer prevention.

Isothiocyanates are versatile cancer-preventive compounds. Evidence from animal studies indicates that the anticarcinogenic activities of ITCs involve all the major stages of tumor growth: initiation, promotion and progression. Epidemiological studies have also shown that dietary intake of ITCs is associated with reduced risk of certain human cancers. A number of mechanisms have been proposed for the chemopreventive activities of ITCs. To identify the molecular targets of ITCs is a first step to understand the molecular mechanisms of ITCs. Studies in recent years have shown that the covalent binding to certain protein targets by ITCs seems to play an important role in ITC-induced apoptosis and cell growth inhibition and other cellular effects. The knowledge gained from these studies may be used to guide future design and screen of better and more efficacious compounds. In this review, we intend to cover all potential protein targets of ITCs so far studied and summarize what are known about their binding sites and the potential biological consequences. In the end, we also offer discussions to shed light onto the relationship between protein binding and reactive oxygen species generation by ITCs.

Sulforaphane activates heat shock response and enhances proteasome activity through up-regulation of Hsp27.

It is conceivable that stimulating proteasome activity for rapid removal of misfolded and oxidized proteins is a promising strategy to prevent and alleviate aging-related diseases. Sulforaphane (SFN), an effective cancer preventive agent derived from cruciferous vegetables, has been shown to enhance proteasome activities in mammalian cells and to reduce the level of oxidized proteins and amyloid ß-induced cytotoxicity. Here, we report that SFN activates heat shock transcription factor 1-mediated heat shock response. Specifically, SFN-induced expression of heat shock protein 27 (Hsp27) underlies SFN-stimulated proteasome activity. SFN-induced proteasome activity was significantly enhanced in Hsp27-overexpressing cells but absent in Hsp27-silenced cells. The role of Hsp27 in regulating proteasome activity was further confirmed in isogenic REG cells, in which SFN-induced proteasome activation was only observed in cells stably overexpressing Hsp27, but not in the Hsp27-free parental cells. Finally, we demonstrated that phosphorylation of Hsp27 is irrelevant to SFN-induced proteasome activation. This study provides a novel mechanism underlying SFN-induced proteasome activity. This is the first report to show that heat shock response by SFN, in addition to the antioxidant response mediated by the Keap1-Nrf2 pathway, may contribute to cytoprotection.

Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice.

Microcystins (MCs), the products of blooming algae Microcystis, are waterborne environmental toxins that have been implicated in the development of liver cancer, necrosis, and even fatal intrahepatic bleeding. Alternative protective approaches in addition to complete removal of MCs in drinking water are urgently needed. In our previous work, we found that sulforaphane (SFN) protects against microcystin-LR (MC-LR)-induced cytotoxicity by activating the NF-E2-related factor 2 (Nrf2)-mediated defensive response in human hepatoma (HepG2) and NIH 3T3 cells. The purpose of this study was to investigate and confirm efficacy the SFN-induced multi-mechanistic defense system against MC-induced hepatotoxicity in an animal model. We report that SFN protected against MC-LR-induced liver damage and animal death at a nontoxic and physiologically relevant dose in BALB/c mice. The protection by SFN included activities of anti-cytochrome P450 induction, anti-oxidation, anti-inflammation, and anti-apoptosis. Our results suggest that SFN may protect mice against MC-induced hepatotoxicity. This raises the possibility of a similar protective effect in human populations, particularly in developing countries where freshwaters are polluted by blooming algae.

Identification of potential protein targets of isothiocyanates by proteomics.

Isothiocyanates (ITCs), such as phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), are effective cancer chemopreventive compounds. It is believed that the major mechanism for the cancer preventive activity of ITCs is through the induction of cell cycle arrest and apoptosis. However, the upstream molecular targets of ITCs have been underexplored until recently. To identify proteins that are covalently modified by ITCs, human non-small cell lung cancer A549 cells were treated with (14)C-PEITC and (14)C-SFN, and the cell lysates were extracted for analysis by 2-D gel electrophoresis and mass spectrometry. After superimposing the colloidal Coomassie blue protein staining pattern with the pattern of radioactivity obtained from X-ray films, it was clear that only a small fraction of cellular proteins contained radioactivity, presumably resulting from selective binding with PEITC or SFN via thiocarbamation. More than 30 proteins with a variety of biological functions were identified with high confidence. Here, we report the identities of these potential ITC target proteins and discuss their biological relevance. The discovery of the protein targets may facilitate studies of the mechanisms by which ITCs exert their cancer preventive activity and provide the molecular basis for designing more efficacious ITC compounds.

Cancer preventive isothiocyanates induce selective degradation of cellular alpha- and beta-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 alpha- and beta-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.

Sulforaphane protects Microcystin-LR-induced toxicity through activation of the Nrf2-mediated defensive response.

Microcystins (MCs), a cyclic heptapeptide hepatotoxins, are mainly produced by the bloom-forming cyanobacerium Microcystis, which has become an environmental hazard worldwide. Long term consumption of MC-contaminated water may induce liver damage, liver cancer, and even human death. Therefore, in addition to removal of MCs in drinking water, novel strategies that prevent health damages are urgently needed. Sulforaphane (SFN), a natural-occurring isothiocyanate from cruciferous vegetables, has been reported to reduce and eliminate toxicities from xenobiotics and carcinogens. The purpose of the present study was to provide mechanistic insights into the SFN-induced antioxidative defense system against MC-LR-induced cytotoxicity. We performed cell viability assays, including MTS assay, colony formation assay and apoptotic cell sorting, to study MC-LR-induced cellular damage and the protective effects by SFN. The results showed that SFN protected MC-LR-induced damages at a nontoxic and physiological relevant dose in HepG2, BRL-3A and NIH 3T3 cells. The protection was Nrf2-mediated as evident by transactivation of Nrf2 and activation of its downstream genes, including NQO1 and HO-1, and elevated intracellular GSH level. Results of our studies indicate that pretreatment of cells with 10muM SFN for 12h significantly protected cells from MC-LR-induced damage. SFN-induced protective response was mediated through Nrf2 pathway.

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 [(14)C]phenethyl ITC and [(14)C]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.

Sensitization of non-small cell lung cancer cells to cisplatin by naturally occurring isothiocyanates.

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.

Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery.

Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic alpha- and beta-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.

Biofunctional coatings via targeted covalent cross-linking of associating triblock proteins.

A method for creating tailorable bioactive surface coatings by targeted cross-linking of network-forming CRC protein polymers is presented. The proteins are triblock constructs composed of two self-associating leucine zipper end domains (C) separated by a soluble, disordered central block (R) containing a cell or molecular binding sequence. The end domains preferentially form trimeric bundles, leading to the formation of a regular, reversible hydrogel network in a wide range of solution conditions. These hydrogel-forming proteins are useful for creating bioactive surface coatings because they self-assemble into networks, physically adsorb to a variety of substrate materials, and can be tailored to display numerous extracellular matrix (ECM)-derived peptides that interact with cells and biological macromolecules. Moreover, due to the close proximity of complementary Glu and Lys residues in the trimeric C bundles, these protein coatings can be stabilized in a targeted manner by covalent cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Here, we demonstrate that such EDC-cross-linked protein coatings are stable in cell culture media and maintain a significant level of biofunctionality when various ECM-derived peptides are embedded in the central soluble block of the proteins. First, we show that EDC cross-linking enables bioinert CRC protein coatings (those without embedded cell binding domains) to resist the adhesion of human foreskin fibroblasts in normal serum medium, but does not impair the ability of cross-linked coatings of CRC-RGDS (proteins with an embedded RGDS integrin binding domain) to promote cellular attachment, focal adhesion formation, and proliferation of these cells. Next, we show that the ability of cross-linked coatings of several new CRC-based proteins containing embedded heparin-binding sequences to bind biotinylated heparin is not significantly impacted over a range of EDC concentrations. The ability to target specific functional groups for covalent cross-linking is made possible by the specificity of protein-protein interactions and represents an important advantage of protein-based materials.

The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells.

Induction of apoptosis underlies a mechanism for inhibiting tumorigenesis by phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). However, the upstream events by which isothiocyanates (ITC) induce apoptosis have not been fully investigated. As electrophiles, ITCs could trigger apoptosis by binding to DNA or proteins or by inducing oxidative stress. To better understand the molecular mechanisms of apoptosis by ITCs, we examined, as a first step, the role of these events in human non-small lung cancer A549 cells. PEITC was a more potent inducer than SFN; it induced apoptosis at 20 micromol/L, whereas SFN induced at 40 micromol/L but not at 20 micromol/L. To study binding with cellular proteins and DNA, cells were treated with (14)C-ITCs; the initial protein binding by PEITC was almost 3-fold than that of SFN. The binding by PEITC increased with time, whereas binding by SFN remained low. Therefore, 4 h after incubation proteins became the predominant targets for PEITC with a 6-fold binding than that of SFN. To characterize the chemical nature of binding by the ITCs, we used bovine serum albumin (BSA) as a surrogate protein. PEITC also modified BSA covalently to a greater extent than SFN occurring exclusively at cysteine residues. Surprisingly, neither PEITC nor SFN bound to DNA or RNA at detectable levels or caused significant DNA strand breakage. The levels of oxidative damage in cells, measured as reactive oxygen species, 8-oxo-deoxyguanosine, and protein carbonyls formation, were greater in cells treated with SFN than PEITC. Because PEITC is a stronger inducer of apoptosis than SFN, these results indicate that direct covalent binding to cellular proteins is an important early event in the induction of apoptosis by the ITCs.

Binding to protein by isothiocyanates: a potential mechanism for apoptosis induction in human non small lung cancer cells.

The upstream events by which isothiocyanates (ITCs) induce apoptosis have not been fully investigated. Numerous studies have reported that the apoptosis was induced by ITCs through generation of reactive oxygen species (ROS) as a result of conjugating with and, consequently, depleting cellular glutathione. As electrophiles, ITCs could potentially trigger apoptosis by binding to macromolecules including DNA and proteins. The results showed that DNA damage may not be an important early event for the apoptosis induction by ITCs. Phenethyl isothiocyanate (PEITC) is a more potent inducer of apoptosis than sulforaphane (SFN) in A549 cells, but SFN induces more ROS generation and oxidative damages than PEITC, suggesting that oxidative stress again is probably not a trigger for apoptosis in these cells. In contrast, we found that PEITC binds more to intracellular proteins than SFN. We identified tubulin as 1 of the protein targets of ITCs through proteomics approach. We showed that the relative tubulin binding affinity of ITCs correlates well with their potency of cell growth inhibition and apoptosis induction. These results collectively suggest that the covalent binding to protein targets, such as tubulin, by ITCs is an important chemical event in apoptosis induction by ITCs in human lung A549 cells.

Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography-tandem mass spectroscopy.

A simple, sensitive and specific LC-MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN-GSH) and N-acetyl cysteine conjugates (SFN-NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extratcion of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN-GSH and SFN-NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC-MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN-GSH, SFN-NAC and SFN were 8.4, 11.0, and 28.2 min,, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01-0.1 microm depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.

A Click Chemistry Approach to Identify Protein Targets of Cancer Chemopreventive Phenethyl Isothiocyanate.

Here we report the identification of protein targets of chemopreventive phenethyl isothiocyanate (PEITC) via "click" chemistry in the A549 human lung cancer cell line, using a novel alkyne-tagged PEITC which was also found to show potent in vitro anticancer activity.

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.