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Background: All known ligands of EGF receptor (EGFR) are characterized by the EGF motif and generated from transmembrane precursors. Results: Prolidase, a cytosolic dipeptidase devoid of EGF motif, binds and activates EGFR independent of its dipeptidase activity when present outside of cell. Conclusion: Prolidase is a novel EGFR ligand.Significance: This shows a new function of prolidase and new mechanism of EGFR activation.

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ErbB2, an important membrane-bound receptor tyrosine kinase, was discovered nearly 30 years ago, but a natural ligand has never been found previously. ErbB2 is also an important oncogene and anticancer target, and its overexpression in cancer is associated with poor disease prognosis. Here, we report that human prolidase (PEPD) is a high affinity ligand of ErbB2 and binds as a homodimer to subdomain 3 in the extracellular domain of this receptor. In ErbB2-overexpressing cells, both ErbB2 monomers and activated dimers exist. PEPD bound to ErbB2 monomers relatively slowly but caused ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. In contrast, PEPD bound rapidly to ErbB2 homodimers and rapidly silenced ErbB2 dimer-Src signaling, a key oncogenic pathway of ErbB2, by disrupting the association of Src with ErbB2. PEPD also caused pronounced ErbB2 depletion, resulting from ErbB2 internalization and degradation. Moreover, PEPD strongly inhibited the DNA synthesis, anchorage-independent growth and invasion and migration of cells that overexpressed ErbB2 but had no effect on cells without ErbB2 overexpression. Cells became sensitized to PEPD upon achieving stable ErbB2 overexpression. Thus, the impact of PEPD on ErbB2 is predominantly inhibitory, and PEPD targets cells addicted to ErbB2. PEPD is also a dipeptidase, but its enzymatic function is not involved in ErbB2 modulation. These findings revise our understanding of ErbB2 and PEPD and may be especially important for combating ErbB2-positive cancers.

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 ...