The Phytochemical Piceatannol Induces the Loss of CBL and CBL-Associated Proteins

Published OnlineFirst March 3, 2009; DOI: 10.1158/1535-7163.MCT-08-0891

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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

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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 Src (kind gift from Dr. J. Michael Chemical Reagents Bishop, University of California at San Francisco), anti- Piceatannol and resveratrol were purchased from two Grb2 mAb (Transduction Labs), and anti-ubiquitin independent suppliers (Sigma and Biomol) to ensure (Zymed). specificity and reproducibility. Compounds 1 to 37 were Reverse Transcription-PCR supplied by Dr. Henrik Hansen and Karm Hans. All new RNA was isolated with TRIzol reagent (Invitrogen) using compounds were identified by nuclear magnetic resonance the manufacturer’s protocol. RNA was then DNase treated and mass spectrometry. Based on the nuclear magnetic with 0.5 units RQ1 RNase-free DNase (Promega) in the resonance spectra, the compounds had a purity of >95%. presence of 20 units RNase Block (Invitrogen) for 30 min at All stilbene analogues were prepared and tested as 37jC. RNA was phenol/chloroform extracted and 2 Ag RNA single isomers. All of the piceatannol-like compounds was used to make cDNA using Superscript II (Invitrogen) were solubilized in DMSO at a stock concentration of following the manufacturer’s protocol. Reverse transcrip- 200 mmol/L and stored as individual aliquots at À20jC tion-PCR was done using the primer drop method (27) until use. Geldanamycin (Alamone Labs), herbimycin A using the Cbl4 (5¶-GCCCTGACCTTCTGATTCCTGCCA-3¶) ¶ ¶ (Life Technologies), curcumin (Sigma), (CBZ-Phe-Arg)2- and MiddleCblF (5 -GATTGATGGCTTCAGGGAAGG-3 ) R110 (Molecular Probes), and epigallocatechin gallate primers to detect c-Cbl/70ZCbl and the mGAPDH-F (Biomol) were made up at stock concentrations in DMSO. (5¶-ACCACAGTCCATGCCATCAC-3¶) and mGAPDH-R Ammonium chloride (BDH/VWR) was dissolved in (5¶-TCCACCACCCTGTTGCTGTA-3¶) primers to detect 10 mmol/L Tris-HCl (pH 7.4) at a stock concentration of GAPDH. 2 mol/L and N-acetylcysteine (Sigma) was dissolved in Flow Cytometric Analysis of Cathepsin Activity PBS at a stock concentration of 100 mmol/L. STI571 was a Mouse fibroblast cells (7.5 Â 105 per well) stably kind gift from Dr. Aru Narendran (Tom Baker Cancer overexpressing 70ZCbl were plated in six-well dishes. Centre). All other chemicals were obtained from Calbio- The following day, cells were treated for 4 h with lysosomal chem and made up at stock concentrations in DMSO. inhibitors and then trypsinized from the plates. Trypsin

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Figure 1. Piceatannol treatment of cells induces Cbl loss. A, Western blots of murine 3T3 fibroblasts stably expressing different Cbl proteins and treated for 3 h with DMSO (À) or 200 Amol/L piceatannol (+). Lysates from vector control fibroblasts were used to detect endogenous c-Cbl, Cbl-b, and actin as a representative loading control. Lysates from cells overexpressing wtCbl, 70ZCbl, p95Cbl, v-Cbl, and D1-355Cbl were probed for Cbl levels to determine which regions of c-Cbl were sensitive to piceatannol treatment. B, Western blots of wtCbl-expressing murine 3T3 fibroblasts treated with 200 Amol/L piceatannol or DMSO and lysed in 1% NP-40 buffer or SDS sample buffer. Both soluble and pellet fractions of the NP-40 lysates are included. The stacking gel was transferred to ensure that Cbl was not being lost due to its inability to enter into the resolving gel. C, Western blots from lysates of 70ZCbl-HA- overexpressing murine 3T3 fibroblast cells treated for 3 h with DMSO (D), 50 Amol/L, or 200 Amol/L piceatannol. Replicate blots were probed using a c-Cbl- specific mAb (Cbl mAb), a c-Cbl-specific polyclonal antibody (Cbl pAb), a HA-specific mAb (HA mAb), or an actin-specific mAb (Actin) as a loading control. D, reverse transcription-PCR using RNA isolated from wtCbl-expressing murine 3T3 fibroblasts treated with 200 Amol/L piceatannol (P)orDMSO (D) for 1, 3, 8, and 24 h. Representative of n z 3, except for B, which is representative of n =2.

was neutralized in 10 mL culture medium and cells 70ZCbl to transform cells, we observed that, unlike other were stained with (CBZ-Phe-Arg)2-R110 and propidium tyrosine kinase inhibitors, piceatannol treatment of 70ZCbl- iodide (Sigma) and analyzed by flow cytometry using the overexpressing murine 3T3 fibroblasts caused the loss of manufacturer’s protocol. Propidium iodide-positive cells Cbl protein (Fig. 1A). We further examined the effect of were subtracted from the (CBZ-Phe-Arg)2-R110-positive piceatannol on endogenous c-Cbl and Cbl-b protein levels cells for the determination of relative cathepsin activity. in murine 3T3 fibroblasts as well as the effect on Cbl The average means of (CBZ-Phe-Arg)2-R110 intensity of protein levels in murine 3T3 fibroblasts overexpressing five experiments were normalized to their respective wtCbl, 70ZCbl, p95Cbl, v-Cbl, and the NH2-terminal untreated controls. Values were then represented as truncation mutant D1-355Cbl. All of the Cbl proteins, relative percentages of normal cathepsin activity. endogenous, wild-type, and mutant, were lost on treatment with piceatannol as shown by Western blotting (Fig. 1A). To ensure that the loss of Cbl was not due to an artifact of Results the lysis or Western blotting procedures, wtCbl-over- Piceatannol Induces the Loss of c-Cbl, Cbl-b, and expressing murine 3T3 fibroblasts were lysed in NP-40 Mutant Oncogenic Cbl Proteins lysis buffer or hot SDS sample buffer and both soluble and Cbl was originally identified based on its homology with pellet fractions of the NP-40 lysates were loaded on a SDS- the retrovirally encoded v-Cbl oncogene (28). In addition, PAGE gel along with the SDS lysates. The stacking gel was we and others have identified two naturally occurring left intact and transferred to nitrocellulose along with the transforming mutant isoforms of c-Cbl: p95Cbl and 70ZCbl resolving gel to ensure that Cbl was not lost due to an (25, 29). These mutant c-Cbl proteins contain deletions of inability to enter into the resolving gel (Fig. 1B). the RING finger, or of areas immediately proximal to the To verify that the observed piceatannol-induced Cbl loss RING finger, which cripple their E3ligase activity (21, 25, was not due to the modification or loss of the epitope 29). These transforming deletion mutations are also recognized by the c-Cbl-specific mAb, Western blots of associated with the constitutive phosphorylation of several HA-tagged 70ZCbl-overexpressing murine 3T3 fibroblasts COOH-terminal tyrosine residues (25, 29). While examin- lysates were reprobed with a polyclonal antibody directed ing the role of tyrosine phosphorylation in the ability of toward the COOH terminus of 70ZCbl as well as a

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monoclonal anti-HA antibody that recognized the HA tag tional level. RNA was isolated from 70ZCbl expressing fused to the NH2 terminus of 70ZCbl. All three antibodies fibroblasts at 1, 3, 8, and 24 h following piceatannol showed that the epitope-tagged 70ZCbl was lost in a treatment. Reverse transcription-PCR for Cbl, normalized dose-responsive manner in cells treated with increasing to the loading control GAPDH, showed no decrease in the concentrations of piceatannol (Fig. 1C). level of Cbl transcript in piceatannol-treated cells (Fig. 1D). The piceatannol-induced loss of Cbl protein levels, while These data indicated that the observed reduction of Cbl visible at time points earlier than 30 min, was most was not due to piceatannol-induced changes in Cbl dramatic after z3h (data not shown). This timeframe for transcription or Cbl transcript stability. Together, these piceatannol-induced loss suggested the possibility that this data suggested that piceatannol is mediating its effect on effect could be at least partially mediated at a transcrip- Cbl at the protein level.

Figure 2. Piceatannol-induced Cbl loss is not due to normal mechanisms of Cbl protein regulation. A, Western blots of lysates and Cbl immunoprecipitations from wtCbl-expressing murine 3T3 fibroblasts transiently transfected with HA-tagged ubiquitin. Cells were treated with 200 Amol/L piceatannol (Pic) for 3 h after a 4 h pretreatment with the proteasomal inhibitor MG132 (25 Amol/L). Cbl was immunoprecipitated from the cell lysates with control IgG or the 7G10 mAb. Western blots were probed for HA-ubiquitin using the 12CA5 anti-HA antibody (left)and reprobed for Cbl using a rabbit polyclonal antibody (right). The stacking gel was left on the Western blot to ensure that Cbl and ubiquitinated Cbl were not being lost due to an inability to enter into the resolving gel. Western blots from lysates of 70ZCbl-expressing murine 3T3 fibroblasts (B) treated with 200 Amol/L piceatannol for 3 h after a 4 h pretreatment with DMSO or the proteasomal inhibitors ALLN (10 Amol/ L), MG132 (25 Amol/L), epoxomycin (200 nmol/L) or (C) treated with the lysosomal inhibitors EST (25 Amol/L), bafilomycin A1 (200 nmol/L), and ammonium chloride (20 mmol/L). Top, probed for Cbl; middle, probed for actin as a loading control. The effectiveness of the proteasomal inhibitors was verified by probing for ubiquitin and the effectiveness of the lysosomal inhibitors was verified by quantifying cathepsin B/L protease activity as a relative percentage of control using the fluorescent cathepsin substrate (CBZ-Phe-Arg)2-R110. D, Western blots from lysates of 70Z/3 cells treated for 6 h with nothing (Ctl), 50 Amol/L piceatannol (Pic), 50 Amol/L H2O2, 100 J UV (UV), or 6 Gy g-radiation (c) to induce apoptosis. Poly(ADP- ribose) polymerase cleavage indicates the induction of apoptosis in the absence of any noticeable Cbl degradation for all treatments except piceatannol. E, Western blots from 70Z/3 cells treated for 6 h with DMSO (À)or50Amol/L piceatannol (+) after pretreatment for 2 h with the indicated concentrations of the caspase inhibitor Boc-D-FMK (C). Dose-dependent decrease in poly(ADP- ribose) polymerase cleavage indicates the effectiveness of the caspase inhibitor. Representative of n z 3, except for A, which is representative of n =2.

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Piceatannol-Induced Cbl Loss Is Not Due to Normal and C). The pharmacologic agents were used at effective Mechanisms of Cbl Protein Regulation doses, as shown by the presence of ubiquitin laddering A distinct smearing of Cbl proteins was often observed (Fig. 2B, bottom) and by the 60% reduction in fluorescence on overexposed Western blots following exposure to of the lysosomal protease substrate (CBZ-Phe-Arg)2-R110 piceatannol (Fig. 2A, right). There is evidence that (Fig. 2C, bottom). c-Cbl is degraded in a ubiquitination-dependent manner Apoptosis-induced caspase activation has been shown to (22–24) and it is conceivable that piceatannol promotes induce the degradation of Cbl (30). To examine whether the this event. To examine whether piceatannol was inducing loss of Cbl was due to the induction of apoptosis, 70Z/3 the ubiquitination of Cbl, wtCbl-overexpressing murine cells were treated with several agents that promote fibroblasts, transfected with HA-tagged ubiquitin, were apoptosis including hydrogen peroxide, UV radiation, pretreated with the proteasome inhibitor MG132 before and g-radiation. All of the agents induced the caspase- piceatannol treatment. By Western blotting with an anti- mediated cleavage of poly(ADP-ribose) polymerase in the HA antibody, HA-ubiquitin was only detected on Cbl 70Z/3cells; however, only piceatannol was able to induce immunoprecipitated from cells treated with MG132 alone the loss of Cbl (Fig. 2D). (Fig. 2A, left). Reprobing these Western blots for Cbl To directly examine the potential role of caspases in (Fig. 2A, right) also showed that MG132 did not induce piceatannol-mediated Cbl loss, 70Z/3cells were treated the same pattern of Cbl smearing as seen after piceatannol with the broad-spectrum caspase inhibitor Boc-D-FMK treatment. We also tested whether inhibition of the before the addition of piceatannol. Although the use proteasome and the lysosome with pharmacologic inhib- of this caspase inhibitor produced a dose-responsive itors could prevent the piceatannol-induced loss of inhibition of piceatannol-induced poly(ADP-ribose) 70ZCbl. Preincubation of 70ZCbl-overexpressing murine polymerase cleavage, it was unable to prevent the fibroblasts with the proteasome inhibitors ALLN, MG132, reduction of Cbl protein levels (Fig. 2E). Taken together, and epoxomycin or the lysosomal inhibitors bafilomycin these data suggested that a previously uncharacterized A1, ammonium chloride, and EST was not sufficient to Cbl-regulatory mechanism was induced on treatment with prevent the piceatannol-induced loss of 70ZCbl (Fig. 2B piceatannol.

Figure 3. Compounds with similar properties as piceatannol are insufficient to induce Cbl loss. A, HA-70ZCbl-expressing murine 3T3 fibroblasts were treated for 3 h with DMSO (D) or several tyrosine and serine/threonine kinase inhibitors including 100 Amol/L (1P) and 200 Amol/L (2P) piceatannol, 10 Amol/L PP1, 10 Amol/L PP2, 10 Amol/L SU6656 (SU), 10 Amol/L AG1296 (1296), 10 Amol/L AG1478 (1478), 2.5 Amol/L geldanamycin (Gel), 1 Amol/L herbimycin A (HA), 50 Amol/L genistein (Gen), 2 Amol/L bisindolylmaleimide I (Bis), 10 Amol/L LY294002 (LY), 25 Amol/L PD98059 (PD), 10 Amol/L U0126 (U), and 10 Amol/L SB203580 (SB). Western blots of lysates were probed for the NH2-terminal HA tag (middle) as well as for the COOH-terminal 7G10 epitope (top). B, Western blots of Cbl immunoprecipitations from K562 cells pretreated for 8 h with 1 Amol/L STI571 and then treated with 200 Amol/L piceatannol (Pic) or DMSO for a further 12 h. Top, probed with an anti-phosphotyrosine antibody; bottom, probed for Cbl. C, 70ZCbl-expressing murine 3T3 fibroblasts were treated for 3 h with DMSO (D), 200 Amol/L piceatannol (P), 200 Amol/L resveratrol (R), 5 mmol/L N-acetylcysteine (N), 50 Amol/L curcumin, or 200 Amol/L epigallocatechin gallate (E). Western blots of cell lysates were probed for Cbl (top) or actin (bottom). Representative of n z 3, except for A, which is representative of n =2.

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Figure 4. Schematic of piceatannol-like molecules screened for the ability to induce Cbl loss. Molecules are labeled by compound number or by abbreviation: piceatannol (Pic), resveratrol (Res), trismethoxy resveratrol (TMR), gallic acid (Gal), (+)-catechin (Cat), and quercetin (Que).

Compounds with Similar Properties as Piceatannol whether kinase inhibition was involved. 70ZCbl-overex- Are Insufficient to Induce Cbl Loss pressing murine fibroblasts were treated with a wide To further confirm that piceatannol was not affecting variety of tyrosine and serine/threonine kinase inhibitors. normal mechanisms of Cbl protein regulation to induce Cbl After 3h treatment, only piceatannol was observed to loss, we next examined the properties attributed to induce a dramatic decrease in amount of Cbl detected piceatannol to see which, if any, were involved in reducing without affecting actin levels (Fig. 3A, lanes 1P and 2P). Cbl protein levels. These properties include the ability of To further confirm this result, we next determined if the piceatannol to inhibit kinases, to induce apoptosis, and to tyrosine phosphorylation status of c-Cbl played a role in act as an antioxidant. As we have already examined and piceatannol-induced Cbl loss. The K562 human erythroleu- excluded the ability of piceatannol to induce apoptosis as a kemia cell line was used to test this possibility, as these cause for Cbl loss (Fig. 2D and E), we next examined cells exhibit Cbl loss on treatment with piceatannol and

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they contain high levels of constitutively tyrosine-phos- and hydrophobicity (Fig. 4). 70Z/3cells were used to phorylated c-Cbl due to the presence of the Bcr-Abl screen the compounds, as these cells have shown the oncogene (29, 31). After 8 h pretreatment with the Abl highest degree of sensitivity to piceatannol-induced Cbl tyrosine kinase inhibitor STI571, c-Cbl tyrosine phosphor- loss. The cells were incubated with 100 Amol/L of each ylation levels in the K562 cells were reduced to undetect- compound for 1 h to avoid artifacts due to the induction of able levels (Fig. 3B, top). A further 12 h treatment of these apoptosis by higher concentrations and later time points, cells with piceatannol resulted in a reduction in the amount and cell lysates were examined for Cbl protein levels by of c-Cbl protein that was comparable with that observed in Western blotting (Fig. 5). Compounds 7, 29, 31, and 33 were cells treated with piceatannol alone (Fig. 3B, bottom). all able to induce Cbl loss. However, only compounds 7, 29, The antioxidant capacity of piceatannol was also exam- and 31 were able to induce the loss of Cbl without affecting ined to determine if it was involved in piceatannol-induced actin protein levels, as was the case with piceatannol Cbl loss. This was accomplished by comparing the (Fig. 5). The common feature of these chemical compounds piceatannol treatment of 70ZCbl-overexpressing murine was the presence of an ortho-catechol ring structure. In fibroblasts with several different antioxidants including piceatannol and in compound 29, the catechol rings are resveratrol, N-acetylcysteine, curcumin, and epigallocate- unprotected, whereas, in compounds 7 and 31, the catechol chin gallate. Western blot analysis indicated that only rings are protected by acetate esters. As cells contain piceatannol was able to induce Cbl loss (Fig. 3C). numerous esterase enzymes, once these compounds Collectively, these data indicate that individual properties enter into cells, they are de-esterified, exposing the catechol commonly associated with piceatannol are not responsible ring. The other common feature of these compounds is for the induction of Cbl loss. that they are all small, highly aromatic molecules Identification of the Structural and Chemical with limited degrees of freedom. Compounds 7 and 29 Requirements for Piceatannol-Induced Cbl Loss are planar trans-stilbenes like piceatannol, whereas Our results indicated that piceatannol was inducing the compound 31 has rotational freedom around its benzylic loss of Cbl in treated cells independently of previously sulfur group. Whereas other compounds in the screen characterized Cbl protein regulatory mechanisms and that possessed these features individually, only piceatannol Cbl loss was due to a heretofore unrecognized property of and these three compounds possessed them in combina- piceatannol. They also indicated that the structurally tion, which suggested that these particular chemical similar compound, resveratrol, was unable to mediate this and structural properties were indispensable for the effect (Fig. 3C). To gain a better understanding of the induction of Cbl loss. mechanism by which piceatannol was inducing Cbl loss, Oxidized Piceatannol Directly Reacts with Purified we screened several piceatannol-like compounds in an Cbl, Leading to the Loss of Cbl Protein attempt to determine the chemical structure(s) required to Due to the requirement for a catechol ring, and our induce Cbl loss. The piceatannol-like molecules used had observation that treatment of cells with piceatannol many chemical and structural similarities to piceatannol and compound 7 led to the medium and cell pellets including aromaticity, size, presence of hydroxyl groups, acquiring a brown color after treatment (data not shown),

Figure 5. Cell-based screen of piceatannol-like molecules for the ability to induce Cbl loss. Western blots of lysates from 70Z/3 cells treated for 3 h with DMSO (D) or 100 Amol/L of the indicated compounds: piceatannol (P), resveratrol (R), trismethoxy resveratrol (T), gallic acid (G), (+)-catechin (C), quercetin (Q), or compound 1-37 (#). Western blots were probed for c-Cbl (top) and actin (bottom). Representative of n =2.

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Figure 6. Oxidized piceatannol directly reacts with purified Cbl, leading to the loss of Cbl protein. A, Cbl immunoprecipitations from 70Z/3 cells were incubated in PBS with combinations of 10 Amol/L H2O2, 20 ng HRP, 200 Amol/L piceatannol, 200 Amol/L resveratrol, and 200 Amol/L stilbene for 15 min at room temperature. Western blots were probed for Cbl with the immunoprecipitating antibody. IgG control represents an immunoprecipitation done using lysis buffer instead of lysates. Bead control represents an immunoprecipitation done without antibody. The heavy chain of the immunoprecipitating antibody is included at the bottom of the blot as a protein control showing the specificity of the degradation. B, Cbl immunoprecipitations from 70Z/3 cells were incubated in PBS with combinations of 10 Amol/L H2O2, 20 ng HRP, 200 Amol/L piceatannol, and 1 mmol/L N-acetylcysteine for 15 min at room temperature. Western blots were probed for Cbl with the immunoprecipitating antibody. C, aqueous solutions of piceatannol, resveratrol, and compound 29 (3,4-dihydroxystilbene) at concentrations of 500 Amol/L were prepared and their A 400 was monitored every 5 s for 150 s. DA 400 values were generated by subtracting these values from the comparable A 400 readings from these compounds in the presence of 10 Amol/L NaIO4 as a source of ROS. DA 400 values were plotted over time to identify the presence of O-benzoquinones, which absorb more strongly at 400 nm. Resveratrol was included as a negative control, as this compound is unable to form an O-benzoquinone due to the positions of its hydroxyl groups. Compound 29 was included as a positive control, as this compound is known to generate O-benzoquinones when oxidized. To confirm the requirement for ROS in the conversion of the piceatannol- like compounds into O-benzoquinones, the same experiment was done in the presence of 1 mmol/L N-acetylcysteine. The presence of this antioxidant completely abrogated the increasing DA 400 over time, indicating that it blocked the formation of O-benzoquinones. Representative of n =3.Points, mean (n = 3); bars, SD (C). we hypothesized that oxidation of the catechol ring into room temperature, the ROS in the reaction mixtures was an O-benzoquinone was involved in piceatannol-induced neutralized by the addition of Laemmli’s buffer containing Cblloss(32,33).Thishypothesiscouldexplaintheh-mercaptoethanol, and the samples were analyzed by observed smearing and loss of Cbl as the result of oxidized Western blotting. Only the combination of piceatannol, piceatannol causing protein cross-linking (34, 35) and H2O2, and HRP was able to induce specific Cbl loss without chemical scission of the peptide backbone (36). To test this affecting the levels of IgG heavy chain from the 7G10 hypothesis, we designed an in vitro assay system to test the antibody used to immunoprecipitate Cbl (Fig. 6A). minimal requirements for piceatannol-induced Cbl loss. To further examine the requirements for piceatannol Immunoprecipitated Cbl was resuspended in PBS and oxidation in piceatannol-induced Cbl loss, N-acetylcysteine aliquoted into several different reaction mixtures contain- was added to the in vitro assay conditions to neutralize ing combinations of trans-stilbene, H2O2, and horseradish ROS produced by the H2O2/HRP system. In the presence of peroxidase (HRP). The H2O2 and HRP were included to this antioxidant, piceatannol-induced Cbl loss was dramat- serve as a source of ROS to promote the oxidation of ically decreased (Fig. 6B), further showing the requirement piceatannol and the other trans-stilbenes. After 15 min at for oxidation of piceatannol in this process.

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To verify that piceatannol and piceatannol-related com- over time, we would be able to determine if these pounds were being converted into O-benzoquinones, we compounds are becoming O-benzoquinones, as this value, examined the spectral properties of these compounds in the the DA400, would increase. Aqueous solutions of piceatan- presence and absence of ROS. O-benzoquinones absorb nol and compound 29 were prepared and their A400 was light at a wavelength of 400 nm much more strongly than monitored every 5 s for 150 s. These values were subtracted do catechols (37); therefore, by subtracting the absorbance from the comparable A400 readings from these compounds A at 400 nm (A400) of an aqueous solution of compound from in the presence of 10 mol/L NaIO4, a source of ROS. Both the A400 of the compound solution in the presence of ROS, piceatannol and compound 29 showed increasing DA400

Figure 7. Piceatannol induces the selective and functional loss of proteins other than Cbl. A to C, murine embryonic fibroblasts, wtCbl-overexpressing murine 3T3 fibroblasts, and 70Z/3 cells, respectively, were treated with DMSO alone (D) or increasing concentrations of piceatannol for 1, 3, and 8 h. Western blots of cell lysates were probed for Cbl, the Cbl interacting proteins c-Abl, PDGFRh, Syk, Src (or the Src family kinase Lyn), and Grb2, and the Cbl noninteracting protein actin. D, immunoprecipitations of PDGFRh, c-Cbl, Lyn, and Src were incubated in PBS with combinations of 10 Amol/L H2O2,20ng HRP, and 50 Amol/L piceatannol for 15 min at room temperature. Western blots of these in vitro reactions were probed for with the specific immunoprecipitating antibodies. E, mouse embryonic fibroblasts were treated with DMSO or 200 Amol/L piceatannol for 8 h or 10 min followed by a 10 min stimulation with 50 ng/mL PDGF-BB. Western blots of cell lysates were probed for PDGFRh, Cbl, phospho-MAPK, and total MAPK. After 8 h pretreatment with piceatannol, PDGFRh levels are reduced only in the Cbl+/+ mouse embryonic fibroblasts, leading to a reduced ability to activate MAPK in response to PDGF-BB treatment. F, A431 cells were pretreated for 3 h with increasing concentrations of piceatannol (32.5, 75, 150, and 300 Amol/L) before a 15 min stimulation with 10 ng/mL EGF. Western blots of cell lysates were probed for EGFR, Cbl, phospho-MAPK, and total MAPK. Representative of n z 3, except for D, which is representative of n =2.

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values over time compared with the negative control this loss was sufficient to affect how cells responded to the resveratrol, indicating the formation of O-benzoquinones PDGFRh ligand, PDGF-BB. To test this, we examined the (Fig. 6C). Resveratrol was included as a negative control, as mitogen-activated protein kinase (MAPK) activation status this compound is unable to form an O-benzoquinone due in murine embryonic fibroblasts that were pretreated with to the positions of its hydroxyl groups (Fig. 4). To confirm piceatannol for 8 h or 10 min before a 10 min stimulation the requirement for ROS in the conversion of the with PDGF-BB (Fig. 7E). MAPK activation was not robustly piceatannol-like compounds into O-benzoquinones, the affected after the 10 min pretreatment, showing that the same experiment was done in the presence of N-acetylcys- activity of piceatannol as a kinase inhibitor does not have a teine. The presence of this antioxidant completely abrogat- major effect on PDGFRh-induced MAPK activation. The 8 h ed the increasing DA400 over time, indicating that it blocked pretreatment with piceatannol, which induced the loss of the formation of O-benzoquinones (Fig. 6C). These data both c-Cbl and PDGFRh, prevented PDGF-BB-induced showed that O-benzoquinones were formed when the ERK1/2 phosphorylation (Fig. 7D). These data showed catechol containing compounds piceatannol and com- that the piceatannol-induced loss of PDGFRh protein also pound 29 were exposed to an oxidant. These experiments induces the loss of PDGFRh function. show that the oxidation of piceatannol and piceatannol-like To examine if piceatannol treatment could be used to compounds induces the formation of O-benzoquinones induce the loss of Cbl-associated oncogenic receptors in a and that the presence of O-benzoquinones is necessary and cancer cell model, the A431 human squamous cell sufficient to induce Cbl loss. carcinoma cell line, which overexpresses epidermal growth Piceatannol Induces the Selective Loss of Proteins factor receptor (EGFR), was treated with increasing OtherThan Cbl concentrations of piceatannol before stimulation with As we were unable to identify a region of Cbl that gave it EGF. Western blots of the A431 cell lysates indicated that, sensitivity to piceatannol (Fig. 1A), it seemed likely that at higher concentrations of piceatannol, both Cbl and EGFR piceatannol could react with many different regions of this protein levels are reduced and that this corresponds to protein. Western blots of piceatannol-treated cells examin- reduced activation of MAPK in response to EGF stimula- ing c-Cbl and the Cbl-associated proteins PDGFRh, c-Abl, tion (Fig. 7F). This result suggested that the piceatannol- c-Src, Lyn, actin, and Grb2 showed that piceatannol was induced loss of Cbl and Cbl-associated signaling proteins also able to induce the loss of other proteins. c-Cbl, may be beneficial as a treatment for a variety of cancers PDGFRh, and c-Abl were all lost as a result of piceatannol promoted by misregulated Cbl-associated PTKs. treatment (Fig. 7A-C). That Lyn, c-Src, Grb2, and action were unaffected by piceatannol treatment also reinforced the observation that there was selectivity to the induction Discussion of protein loss of piceatannol (Fig. 7A-C). In addition to Piceatannol is a naturally occurring small molecule known the selective loss of proteins in vivo, Lyn shifted to a lower for its kinase inhibition and anticancer properties (1, 6–11). apparent molecular weight after piceatannol treatment We have shown that piceatannol is also able to induce the (Fig. 7C, 70Z/3). This molecular weight shift is reminiscent loss of Cbl proteins (Fig. 1). By screening a small library of of a change in the tyrosine phosphorylation status of the piceatannol-like compounds for their ability to induce Cbl protein and was likely the result of piceatannol-induced loss in cells, we determined that the Cbl loss is associated kinase inhibition. with the catechol ring structure of piceatannol (Figs. 4 To better understand the protein specificity for piceatan- and 5). The results of this screen showed that only 3of 19 nol-induced protein loss observed in piceatannol-treated ortho-catechol-containing compounds, compounds 7, 29, cells, we tested several different immunoprecipitated and 31, are able to induce Cbl loss to a similar extent as proteins in our in vitro reaction with piceatannol and/or piceatannol (Fig. 5), indicating that, in addition to the ROS. We hypothesized that in vitro most proteins would be catechol ring, there are further chemical requirements sensitive to piceatannol-induced protein loss, considering for Cbl loss. Catechol-containing compounds lacking an that we have been unable to identify a specific region of Cbl aromatic substituent, including compounds 12 and 15, that led to its sensitivity to piceatannol (Fig. 1A) and that were unable to induce Cbl loss (Fig. 4), indicating that the there are many different amino acids with nucleophilic presence of an aromatic substituent was also important to atoms in their side chains, none of which are unique to Cbl, the process. which could react with O-benzoquinones including cyste- These data suggest a mechanism of action in which ine, methionine, histidine, tryptophan, serine, tyrosine, piceatannol enters into cells and is subsequently oxidized lysine, arginine, and proline (38). Western blotting of into an electrophilic O-benzoquinone that readily reacts in vitro reactions containing c-Cbl, c-Src, Lyn, or PDGFRh with nucleophilic atoms from the amino acid side chains of confirmed that, similar to c-Cbl, all of these proteins were Cbl (38). This would then lead to the cross-linking of Cbl, lost in the presence of piceatannol and ROS (Fig. 7D). These explaining the smearing observed by Western blotting data indicated that piceatannol-induced protein loss had a after piceatannol treatment (refs. 34, 35; Fig. 2A, right). The broader specificity in vitro than in cell culture. O-benzoquinones can also react with peptide bonds, Based on our observation that piceatannol treatment led inducing scission of the polypeptide backbone (36) and to a loss of PDGFRh, we were interested in determining if leading to the loss of Cbl observed after piceatannol

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treatment. Further supporting this plausible mechanism of aptly viewed as a marker for piceatannol activity. This is action, using an in vitro reaction that recapitulates the illustrated by the effects of piceatannol on RTK protein minimum requirements for piceatannol-induced Cbl loss, levels and signaling (Fig. 7). Whereas the loss of Cbl has we show that this phenomenon is directly mediated been associated with an increase in RTK-induced MAPK through the oxidative conversion of piceatannol into an activation, piceatannol treatment induced the loss of Cbl, O-benzoquinone (Fig. 6). In addition, similar observations the loss of RTKs, and a decrease in RTK-induced MAPK and similar mechanisms of action have been shown for signaling (Fig. 7E and F). It is also illustrated by electrophilic a,h-unsaturated ketones, such as curcumin piceatannol-induced apoptosis. Cellular sensitivity to and acrolein (39, 40). These and other a,h-unsaturated piceatannol-induced Cbl-loss correlates with the sensitivity ketones have been shown to covalently modify and to piceatannol-induced apoptosis in several different cell cross-link proteins such as nuclear factor-nB (39), lines (data not shown). However, these effects were p53(41), peroxisome proliferator-activated receptor g (42), independent of the isoform of Cbl present. Logically, the SNAP-25, and VAMP (40). As O-benzoquinones contain loss of a transforming Cbl mutant should make cells an a,h-unsaturated ketone, these observations support more susceptible to apoptosis and the loss wtCbl, a the requirement for the oxidation of piceatannol to induce negative regulator of cell growth and survival, would Cbl loss. promote cell survival. This suggests that a common Similar to these other a,h-unsaturated ketones, piceatan- piceatannol-sensitive element between the cell lines was nol also showed a limited protein specificity in cell culture. responsible for the induction of apoptosis and also suggests In addition to inducing the loss of Cbl, piceatannol that piceatannol-induced Cbl loss is an effective marker of treatment of cells induced the selective loss of three Cbl- the biological activity of piceatannol. associated proteins: PDGFRh, c-Abl, and EGFR (Fig. 7). The ability of piceatannol to induce the loss of specific Other proteins were unaffected in piceatannol-treated cells signaling proteins has implications for its use in the including c-Src, Lyn, Syk, actin, and Grb2. This evidence treatment of disease. The piceatannol-induced loss of RTK- suggests that piceatannol is associating specifically with mediated MAPK activation (Fig. 7E and F) and the structure(s), within cells that have restrictive binding piceatannol-induced loss of Cbl (Fig. 1) may be effective requirements, because not all aromatic catechol-containing for the treatment of cancers in which these piceatannol- compounds were able to mediate protein loss. However, sensitive proteins are involved. In addition, the inhibition of other experimental data argue against such a direct multiple PTKs, which may be possible through the use and specific interaction. (a) The absence of a specific of piceatannol as a therapeutic, has proven to be more domain of Cbl that is responsible for its sensitivity to effective as a cancer treatment than the inhibition of single piceatannol (Fig. 1A). (b) Syk and Src family kinases, PTKs (43–45). There also exists the possibility for synergy proteins known to be functionally inhibited by piceatannol between piceatannol and several current therapeutics that and thus assumed to physically associate with piceatannol are known to produce ROS (46–50). Although piceatannol (9, 10), are not affected by piceatannol-induced protein loss has been studied as an anticancer agent, a lack of mechanistic in cell culture experiments (Fig. 7A-C). insight has prevented any further development. Our Other factors affecting specificity of protein loss may methods and our data identify the active chemical structures reside within the microenvironment of the cell including involved in piceatannol-induced protein loss providing the local concentrations of ROS, which are required to a platform from which more effective piceatannol-like activate piceatannol, the local concentrations of glutathione compounds can be designed and tested (Figs. 4 and 5). and other cellular antioxidants, and the compartmentaliza- We have made the observation that piceatannol treat- tion of the specific proteins of interest. This is supported by ment of cells causes a reduction in protein levels of Cbl and the actions of compound 33, which, unlike piceatannol, specific Cbl-associated proteins. This work suggests a already contains an O-benzoquinone structure (Fig. 4). This mechanism of piceatannol action in which oxidized compound induced nonspecific protein loss (Fig. 5), piceatannol reacts with nucleophilic atoms from the amino suggesting that the location of catechol oxidation is acid side chains of Cbl, leading to protein cross-linking, and involved in the selectivity for the protein loss reaction. reacts with the peptide backbone of Cbl, inducing protein Another explanation for the lack of specificity of piceatan- loss. The ability of piceatannol to induce protein loss also nol in vitro may be due to the nature of the in vitro has important functional consequences with respect to its experimental conditions. The immunoprecipitation of use as a kinase inhibitor: if piceatannol is to be used as a proteins in NP-40 lysis buffer may alter their conformation specific kinase inhibitor, it must be used at low concen- such that reactive residues previously hidden become trations for short treatment periods to avoid the potentially exposed and available to react with oxidized piceatannol. overlapping effects of Cbl and Cbl-associated protein loss. Considering that several signaling molecules are affected Cbl and its associated proteins are involved in many of the by piceatannol-induced protein loss (Fig. 7) and that same signaling pathways as Syk and Src family kinases, several others have their catalytic activity inhibited by and as such, this work also offers a strong cautionary note piceatannol (Fig. 7C), it is unlikely that Cbl loss is on the interpretation of the involvement of these PTKs specifically responsible for all of the biological activities when relying exclusively on the use of piceatannol as a of piceatannol. Piceatannol-induced protein loss is more tyrosine kinase inhibitor.

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The phytochemical piceatannol induces the loss of CBL and CBL-associated proteins

Alexander C. Klimowicz, Sabine A. Bisson, Karm Hans, et al.

Mol Cancer Ther 2009;8:602-614. Published OnlineFirst March 3, 2009.

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