Published OnlineFirst March 3, 2009; DOI: 10.1158/1535-7163.MCT-08-0891 602 The phytochemical piceatannol induces the loss of CBL and CBL-associated proteins Alexander C. Klimowicz,1 Sabine A. Bisson,1 signaling pathways commonly associated with cancer. Karm Hans,2 Elizabeth M. Long,1 This work uncovers a new, piceatannol-dependent effect Henrik C. Hansen,2 and Stephen M. Robbins1 and shows a novel way in which this phenomenon can be exploited to inhibit disease-associated signaling pathways. 1Southern Alberta Cancer Research Institute, Departments of [Mol Cancer Ther 2009;8(3):602–14] Oncology and Biochemistry and Molecular Biology, and 2Department of Chemistry, The University of Calgary, Calgary, Alberta, Canada Introduction Piceatannol is a small molecule that was initially isolated as Abstract the antileukemic agent from the domesticated oilseed Piceatannol is a naturally occurring bioactive stilbene with Euphorbia lagascae (1). In mammals and mammalian cell documented antileukemic properties. It has been exten- culture, piceatannol has also been shown to have beneficial sively used as a Syk-selective protein tyrosine kinase effects as an anti-inflammatory agent (2, 3), an anti- inhibitor for the study of various signaling pathways. histamine (4, 5), and a general anticancer agent (1, 6–8). Herein, we show that the hydroxystilbene, piceatannol, Piceatannol is thought to accomplish these varied effects and related catechol ring-containing compounds are able through its inhibition of specific tyrosine and serine/ to induce the loss of the Cbl family of proteins. Normal threonine protein kinases. Piceatannol was initially shown cellular Cbl-regulatory mechanisms were not involved in to be, and is still commonly used as, a Syk-selective this process. Screening of a small library of piceatannol- protein tyrosine kinase (PTK) inhibitor (9). It can also like compounds indicated that aromaticity and a catechol inhibit other tyrosine kinases including Src, Lck, and FAK, ring were required for the induction of Cbl loss. Further albeit with lower efficiency (9–11). More recently, picea- examination of these two chemical properties showed that tannol has been shown to inhibit several serine/threonine the oxidative conversion of the catechol ring of piceatan- kinases (12, 13). The cellular effects of piceatannol are not nol into a highly reactive O-benzoquinone was the cause limited to kinase inhibition; it has been shown to induce of piceatannol-induced Cbl loss. Characterization of the apoptosis, which may be related to its inhibition of Cbl selectivity of piceatannol-induced protein loss revealed mitochondrial F0F1-ATPase activity (14, 15), and to induce that this compound was also able to induce the functional DNA damage (16–18). Piceatannol has also been shown to loss of specific Cbl-associated proteins involved in have antioxidant properties (19, 20). Despite the extensive characterization of its properties and cellular effects, piceatannol has never been associated with the loss of specific proteins. Herein, we show that piceatannol induces the loss of the Cbl family of proteins Received 9/12/08; revised 12/18/08; accepted 12/31/08; and specific Cbl-associated PTKs in a reactive oxygen published OnlineFirst 3/3/09. species (ROS)-dependent manner. Grant support: Canadian Institutes of Health Research and Alberta Cancer Board; Alberta Heritage Foundation for Medical Research, Province of The c-Cbl proto-oncogene is an adaptor protein and a Alberta, and R&D Health Research Foundation graduate fellowships (A.C. RING finger E3ubiquitin ligase; as such, it acts as both Klimowicz); and Alberta Heritage Foundation for Medical Research and Killam positive and negative regulators of many PTKs. On Scholarship at the University of Calgary graduate fellowship (S.A. Bisson). activation of receptor tyrosine kinases (RTK), c-Cbl is The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked recruited from the cytosol to the activated receptor complex advertisement in accordance with 18 U.S.C. Section 1734 solely to where it recruits signaling effector proteins and positively indicate this fact. regulates signaling by acting as an adaptor protein. Cbl Note: S.M. Robbins currently holds a Canada Research Chair in Cancer then ubiquitinates these receptors as well as many of their Biology and is a Scientist of the Alberta Heritage Foundation for Medical Research. associated signaling proteins (21), causing them to be Current address for S.A. Bisson: Howard Hughes Medical Institute, degraded by the proteasome and/or lysosome (21). Cbl Department of Microbiology and Immunology, University of California at itself is also regulated by ubiquitin-mediated proteolysis San Francisco, 513 Parnassus Avenue, HSW 1501, San Francisco, CA 94143-0552. Current address for H.C. Hansen: Resverlogix, 279 Midpark (21–24). Thus, through its E3ubiquitin ligase activity, Cbl Way Southeast, Suite 202, Calgary, Alberta, Canada T2X 1M2. also functions as a potent negative regulator of PTK Requests for reprints: Stephen M. Robbins, Southern Alberta Cancer signaling. Research Institute, Departments of Oncology and Biochemistry and We have found that piceatannol dramatically reduces Molecular Biology, The University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada T2N-4N1. Phone: 403-220-4304; cellular Cbl protein levels independently of the protea- Fax: 403-283-8727; [email protected] some, the lysosome, and caspase activation. By screening a Copyright C 2009 American Association for Cancer Research. small library of piceatannol-like compounds, we deter- doi:10.1158/1535-7163.MCT-08-0891 mined that compound-induced Cbl protein loss was Mol Cancer Ther 2009;8(3). March 2009 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst March 3, 2009; DOI: 10.1158/1535-7163.MCT-08-0891 Molecular Cancer Therapeutics 603 mediated directly by aromatic compounds that contain Cell Stimulations and Transfections catechol rings through their oxidative conversion into a Murine 3T3 fibroblasts ectopically expressing Cbl, mouse highly reactive O-benzoquinone. We further characterized embryonic fibroblasts, and A431 cells were plated in six- the protein selectivity of this process and established the well plates with 5 Â 105 per well in 2 mL supplemented applicability of these observations to cancer treatment. This DMEM and grown overnight before their use in experi- work characterizes a previously unrecognized piceatannol- ments. K562 and 70Z/3cells were resuspended at 8 Â dependent effect and shows a novel way in which this 105/mL in complete RPMI in a total volume of 4 mL in effect can be exploited as a therapeutic to inhibit disease- 5 mL round-bottomed polystyrene tubes the day they associated signaling pathways. were used in experiments. Six-well plates with 5 Â 105 Cbl- expressing 3T3 cells per well were grown in 2 mL supplemented DMEM overnight before transfection. Trans- Materials and Methods fection with pMT/HA-ubiquitin, a kind gift of Dr. Jane Cell Lines McGlade (Hospital for Sick Children, Toronto, Ontario, Murine 3T3 fibroblasts overexpressing wild-type c-Cbl Canada), was accomplished using Fugene 6 (Roche) accord- (wtCbl), 70ZCbl, and p95Cbl were generated and main- ing to the manufacturer’s protocol. Twenty-four hours after tained as described previously (25). The NH2-terminal transfection, cells were treated with MG132 and/or picea- HA-tagged v-Cbl allele was generously provided by tannol. The specific details regarding each experiment are Dr. Wallace Langdon and cloned into pBabepuro3as included in the figure legends. At the indicated times, cells described previously for p95Cbl (25). The Cbl deletion were lysed in NP-40 lysis buffer as described previously (25). Â mutant D1-355Cbl, a c-Cbl NH2-terminal truncation mutant Alternatively, cells were lysed in hot 2 SDS Laemmli’s that begins at codon 356, was generated by PCR using sample buffer after being washed in PBS. pBabe/wtCbl(N) and a NH2-terminal primer introducing a Immunoprecipitations and Western Blotting XhoI site in front of c-Cbl codon 356 [primer XhoCbl356(s) Immunoprecipitations and Western blots were done as 5¶-CCTCGAGACTCCCCAAGACCATATC-3¶ and primer described previously (25) using the following antibodies: Cbl6(a) 5¶-TCTCTGGAGGGACAGTCGC-3¶]. This PCR 7G10 monoclonal antibody (mAb) against c-Cbl (Upstate), product was digested with XhoI and BglII and ligated rabbit anti-Cbl polyclonal antibody (Santa Cruz Biotech- into XhoI/BglII-digested pBabe/wtCbl(N). The resultant nology), 12CA5 mAb against HA, anti-actin mAb1501 plasmid, pBabe/D1-355Cbl, was stably expressed in mu- (Chemicon), C2-10 mAb against poly(ADP-ribose) poly- rine 3T3 fibroblasts as above (25). The mouse embryonic merase (Trevigen), rabbit anti-platelet-derived growth fibroblasts were kindly provided by Dr. Hamid Band (26). factor receptor (PDGFR) polyclonal antibody (Santa Cruz The mouse embryonic fibroblasts and the A431 cells were Biotechnology), rabbit anti-Syk (Santa Cruz Biotechnology), maintained similar to the 3T3 fibroblasts in the absence of rabbit anti-p56Lyn polyclonal antibody (kind gift from Dr. any selection. The 70Z/3murine pre-B-cell line and the Anthony L. DeFranco, University of California at San K562 human erythroleukemia cell line were maintained as Francisco), rabbit anti-Abl (Cell Signaling Technology), described previously (25). Src327 mAb against