Lixin Mi

Covalent Binding to Tubulin by Isothiocyanates A MECHANISM OF CELL GROWTH ARREST AND APOPTOSIS

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.

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.

Cancer Preventive Isothiocyanates Induce Selective Degradation of Cellular α- and β-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.

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 β-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.

Selective Depletion of Mutant p53 by Cancer Chemopreventive Isothiocyanates and Their Structure−Activity Relationships

Isothiocyanates (ITCs) derived from cruciferous vegetables induce apoptosis in cancer cells. We demonstrate that certain naturally occurring ITCs selectively deplete mutant p53 but not the wild-type and do so via a transcription-independent mechanism. Direct p53 binding followed by conformational changes appears to be a mechanism by which mutant p53 is depleted. Structure-activity relationship studies (SARs) using naturally occurring and synthetic ITCs show that depletion is influenced by the ITC side-chain moiety. Furthermore, we show that cells with p53 mutations are more sensitive to cytotoxicity induced by phenethyl isothiocyanate (PEITC) than those with the wild-type protein. 2,2-Diphenylethyl ITC, a synthetic ITC, is one of the most potent depletors of mutant p53 studies and induces apoptosis to the greatest extent in mutant p53 breast cancer cells. Collectively, this study shows that mutant p53 depletion may be an important novel target for cancer chemoprevention and therapy by natural and synthetic ITCs.

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.

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.

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.

Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin

SCOPE Naturally-occurring chemopreventive agent phenethyl isothiocyanate (PEITC), derived primarily from watercress, has been shown to inhibit cell growth and induce apoptosis in cancer cells. In this study, we examined the potential of PEITC in enhancing cisplatin-induced apoptosis in cervical cancer cells and its mechanisms. METHODS AND RESULTS HeLa cells were exposed to PEITC, cisplatin or both. Pretreatment of cells with PEITC strongly enhanced cisplatin-induced cytotoxicity. PEITC activated the mitogen-activated protein kinases, including c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38. Caspase-3 activity assay demonstrated that the synergistic induction of apoptosis was significantly attenuated by MEK1/2 inhibitor U0126, but not by JNK or p38 inhibitor, suggesting that ERK activation is responsible for the synergistic effect. We found that NF-κB signaling pathway is not involved in the synergistic effect. Sulforaphane and benzyl isothiocyanate, two other members of the isothiocyanate family, also sensitize HeLa cells to apoptosis induced by cisplatin. Furthermore, we found that the synergistic effect was also observed in cervical cancer C33A and breast cancer MCF-7 cells but not in normal mammary epithelial MCF-10A cells. Finally, we demonstrated that Noxa induction was associated with apoptosis induced by PEITC plus cisplatin. CONCLUSION Taken together, this study shows that PEITC can sensitize cancer cells to apoptosis induced by cisplatin and this effect is mediated through ERK activation, suggesting the potential of PEITC to be used as an adjuvant with cisplatin in combination therapeutic treatments.

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.