Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of β-tubulin at Cys 12 and suppression of microtubule dynamics

Xu, Wanhong; Xi, Biao; Wu, Jieying; An, Hongyu; Zhu, Jenny; Abassi, Yama; Feinstein, Stuart C.; Gaylord, Michelle; Geng, Bao-qin; Yan, Huifang; Fan, Weimin; Sui, Meihua; Wang, Xiaobo; Xu, Xiao Yun

doi:10.1158/1535-7163.mct-09-0548

Cited by 23 publications

(13 citation statements)

References 37 publications

(61 reference statements)

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“…Cys12β is involved in GTP binding, as indicated by photoaffinity labeling experiments [39]. Our finding that Cys12β is succinated when fumarate levels are elevated raises the interesting possibility that succination could affect GTP binding and thus interfere with the normal microtubule dynamics, as recently shown for the natural product derivative bis(4-fluorobenzyl)trisulfide that also binds to Cys-12β [40]. …”

Section: Discussionsupporting

confidence: 53%

Identification of protein succination as a novel modification of tubulin

Piroli

Manuel

Walla

et al. 2014

Biochemical Journal

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Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). We demonstrate that both alpha (α) and beta (β) tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound dimethylfumarate (DMF, 500 μM) inhibited polymerization up to 35% and 59%, respectively. Using mass spectrometry we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteines increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose vs. normal glucose also had reduced reactivity with the anti-αtubulin antibody; suggesting that succination may interfere with tubulin:protein interactions. DMF reacted rapidly with 11 of the 20 cysteines in the αβ tubulin dimer, decreased the number of free sulfhydryls and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggests that inhibition of tubulin polymerization is an important, undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics.

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Section: Discussionsupporting

confidence: 53%

Identification of protein succination as a novel modification of tubulin

Piroli

Manuel

Walla

et al. 2014

Biochemical Journal

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“…In the current study, we investigated the factors involved in the significant metabolism of BFBTS in rat blood and identified the metabolites of BFBTS. As indicated in the literature (Hosono et al, 2005;Xu et al, 2009), the trisulfide might interact with the cysteine residues in biologic macromolecules, thus we confirmed the interactions between BFBTS and hemoglobin, the major protein in red blood cells. The results will contribute to a better understanding of the metabolic mechanism of BFBTS and other trisulfides in blood.…”

Section: Introductionsupporting

confidence: 87%

Metabolism of Bis(4-fluorobenzyl)trisulfide and Its Formation of Hemoglobin Adduct in Rat Erythrocytes

Pān

Sun

et al. 2013

Drug Metab Dispos

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Bis(4-fluorobenzyl)trisulfide (BFBTS) is a promising new antitumor agent under investigation. It was metabolized rapidly in vivo in rat, but the metabolic fate and primary site of metabolism have not been clarified. In this study, we investigated the role of blood in the metabolism of BFBTS and compared the BFBTS metabolic potencies in whole blood, plasma, and red blood cells (RBCs) in vitro. Three major metabolites of BFBTS [bis(4-fluorobenzyl) disulfide, para-fluorobenzyl-mercaptan, and para-fluorobenzoic acid] were detected in RBCs and whole blood. Significant metabolism of BFBTS was observed in RBCs that were identified as the primary site of BFBTS metabolism. Thiols, including endogenous thiols and hemoglobin, were proven to be the critical factor in BFBTS metabolism. S-Fluorobenzylmercaptocysteine Hb (hemoglobin) adducts were characterized in vitro at BFBTS concentration of 250 mM and higher, whereas such Hb adducts were not detected in RBCs from Sprague-Dawley rats receiving a single intravenous injection of BFBTS at a high dose of 50 mg/kg. Liquid chromatography-tandem mass spectrometry results revealed that adduction induced by BFBTS was prone to take place at Cys125 of globin b chains. Otherwise, glutathionylation of Hb was also observed that may be attributed to the oxidative effect of BFBTS. In summary, BFBTS was unstable when it met with thiols, and RBCs were the main site of BFBTS metabolism. Hb adducts induced by BFBTS could be detected in vitro at high concentration but not in vivo even at high dose.

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“…This is consistent with the knowledge that the -NϭCϭS group of AITC is electrophilic and readily reacts with cysteine thiols, but the reaction is reversible (1). Although it remains to be determined if AITC binding to the cysteine residues or a specific cysteine residue of the tubulins is responsible for increased ubiquitination, most of the ␤-tubulin cysteine residues targeted by AITC have also been shown to be targeted by a number of antitumor agents, including Cys-12 by bis(4-fluorobenzyl)trisulfide and arsenic (19,20), Cys-239 by 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (21), and Cys-239 and Cys-354 by colchicine and colchicinoids (22,23). Moreover, mutagenesis of Cys-12 or Cys-354 in ␤-tubulin was either lethal or significantly affected the structure and function of the tubulin in Saccharomyces cerevisiae (24).…”

Section: Discussionmentioning

confidence: 99%

Allyl Isothiocyanate Arrests Cancer Cells in Mitosis, and Mitotic Arrest in Turn Leads to Apoptosis via Bcl-2 Protein Phosphorylation

Geng¹,

Tang²,

Li³

et al. 2011

Journal of Biological Chemistry

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Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of ␣-and ␤-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITCinduced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G 1 phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet.Allyl isothiocyanate (AITC) 2 is a naturally occurring compound that possesses both antimicrobial and anticancer activities. Many commonly consumed cruciferous vegetables are rich sources of AITC, such as mustard, horseradish, wasabi, and cabbage. Its bactericidal and fungicidal activities were demonstrated against a variety of pathogens, and its anticancer activities were shown in both cultured cancer cell lines and animal tumor models (1). Bioavailability of AITC is extremely high; nearly 90% of orally administered AITC is absorbed (1). Although available evidence indicates that the anticancer activity of AITC is neither cell-nor tissue-specific, we have recently shown that AITC is selectively delivered to bladder tissue through urinary excretion and potently inhibits cancer development and muscle invasion in an orthotopic rat bladder cancer model (2). Moreover, an AITC-rich mustard seed powder also strongly inhibited bladder cancer development and muscle invasion in vivo (3). Thus, AITC is highly promising for bladder cancer prevention and/or treatment. These results are also consistent with epidemiological studies showing an inverse association between consumption of cruciferous vegetables and bladder cancer risk (4, 5). In light of these findings, this study focuses on human bladder cancer cells.Previous studies have shown that AITC causes cell cycle arrest and apoptosis in cancer cell lines of different tissue origins in vitro and in several tumor xenograft models in vivo, and it modulates many genes and proteins involved in cancer cell survival and proliferation (1). In our recent studies, both AITC and the AITC-rich mustard seed powder mentioned above arrested bladder cancer cells in G 2 /M ...

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Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of β-tubulin at Cys 12 and suppression of microtubule dynamics

Cited by 23 publications

References 37 publications

Identification of protein succination as a novel modification of tubulin

Identification of protein succination as a novel modification of tubulin

Metabolism of Bis(4-fluorobenzyl)trisulfide and Its Formation of Hemoglobin Adduct in Rat Erythrocytes

Allyl Isothiocyanate Arrests Cancer Cells in Mitosis, and Mitotic Arrest in Turn Leads to Apoptosis via Bcl-2 Protein Phosphorylation

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