Corrections

MEDICAL SCIENCESCorrection for “Benzoquinone ansamycin 17AAG binds to mi-tochondrial voltage-dependent anion channel and inhibits cellinvasion,” by Qian Xie, Robert Wondergem, Yuehai Shen, GregCavey, Jiyuan Ke, Ryan Thompson, Robert Bradley, JenniferDaughtery-Holtrop, Yong Xu, Edwin Chen, Hanan Omar,Neal Rosen, David Wenkert, H. Eric Xu, and George F. VandeWoude, which appeared in issue 10, March 8, 2011, of Proc NatlAcad Sci USA (108:4105–4110; first published February 22, 2011;doi:10.1073/pnas.1015181108).The authors note that the author name Jennifer Daughtery-

Holtrop should have appeared as Jennifer Daugherty-Holtrop.The corrected author line appears below. The online version hasbeen corrected.

Qian Xiea, Robert Wondergemb, Yuehai Shenc, GregCaveyd, Jiyuan Kee, Ryan Thompsona, Robert Bradleya,Jennifer Daugherty-Holtrope, Yong Xue, Edwin Chenc,Hanan Omarc, Neal Rosenf, David Wenkertc, H. Eric Xue,and George F. Vande Woudea,1

www.pnas.org/cgi/doi/10.1073/pnas.1103553108

APPLIED BIOLOGICAL SCIENCESCorrection for “Quantitative selection of DNA aptamers throughmicrofluidic selection and high-throughput sequencing,” byMinseon Cho, Yi Xiao, Jeff Nie, Ron Stewart, AndrewT. Csordas,Seung Soo Oh, James A. Thomson, and H. Tom Soh, which ap-peared in issue 35, August 31, 2010, of Proc Natl Acad Sci USA(107:15373–15378; first published August 12, 2010; 10.1073/pnas.1009331107).The authors note the following statement should be added to

the Acknowledgments: “Financial support for M.C. was also par-tially provided by a Postdoctoral Fellowship from the CaliforniaInstitute for Regenerative Medicine (CIRM).”

www.pnas.org/cgi/doi/10.1073/pnas.1103208108

5472 | PNAS | March 29, 2011 | vol. 108 | no. 13 www.pnas.org

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Benzoquinone ansamycin 17AAG binds tomitochondrial voltage-dependent anionchannel and inhibits cell invasionQian Xiea, Robert Wondergemb, Yuehai Shenc, Greg Caveyd, Jiyuan Kee, Ryan Thompsona, Robert Bradleya,Jennifer Daugherty-Holtrope, Yong Xue, Edwin Chenc, Hanan Omarc, Neal Rosenf, David Wenkertc, H. Eric Xue,and George F. Vande Woudea,1

aLaboratory of Molecular Oncology, Van Andel Research Institute, Grand Rapids, MI 49503; bDepartment of Physiology, East Tennessee State University,Johnson City, TN 37614; cDepartment of Physiology, Michigan State University, East Lansing, MI 48824; dMass Spectrometry and Proteomics Laboratory,Van Andel Research Institute, Grand Rapids, MI 49503; eLaboratory of Structure Science, Van Andel Research Institute, Grand Rapids, MI 49503; andfProgram in Molecular Pharmacology and Chemistry, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065

Edited* by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved January 12, 2011 (received for review October 22, 2010)

Geldanamycin and its derivative 17AAG [17-(Allylamino)-17-deme-thoxygeldanamycin, telatinib] bind selectively to the Hsp90 chap-erone protein and inhibit its function. We discovered that thesedrugs associate with mitochondria, specifically to the mitochon-drial membrane voltage-dependent anion channel (VDAC) viaa hydrophobic interaction that is independent of HSP90. In vitro,17AAG functions as a Ca2+ mitochondrial regulator similar to ben-zoquinone-ubiquinones like Ub0. All of these compounds increaseintracellular Ca2+ and diminish the plasma membrane cationic cur-rent, inhibiting urokinase activity and cell invasion. In contrast, theHSP90 inhibitor radicicol, lacking a bezoquinone moiety, has nomeasurable effect on cationic current and is less effective in influ-encing intercellular Ca2+ concentration. We conclude that some ofthe effects of 17-AAG and other ansamycins are due to their effectson VDAC and that this may play a role in their clinical activity.

benzoquinone ansamycin | calcium regulation

Ansamycin antibiotics, such as Geldanamycin (GA) and itsderivative 17AAG, bind to a conserved pocket in the ami-

noterminal portion of the Hsp90 chaperone protein and inhibitits function. This results in the degradation of Hsp90 clientproteins, which include key components of mitogenic signalingpathways and several oncoproteins (1). 17-(Allylamino)-17-demethoxygeldanamycin (17AAG, telatinib) effectively inducesdegradation of Hsp90 client proteins in vivo and at nontoxicdoses, has antitumor activity in a wide variety of murine xeno-graft and genetically engineered tumor models, and has beenshown to have significant clinical activity in patients with HER2breast cancer and adult refractory AML (2, 3).17AAG and all GA derivatives have quinone moieties that are

metabolized by NAD(P)H: quinone oxidoreductase1 (NQO1)(4, 5). Kelland et al. (6) first reported the relationship betweenlevels ofNQO1 and the sensitivity to 17AAG.Others have reportedthat, in vitro, via quinone catalytic activity, GA leads to superoxideformation without affecting HSP90 (7), suggesting that 17AAGfunctions through mitochondria. Here, we report that 17AAG candirectly bind to mitochondria, specifically to the voltage-dependentanion channel (VDAC) through hydrophobic interaction.We showthat HSP90 competes with mitochondria for binding with 17AAG.Moreover, GA derivatives have effects similar to ubiquinone, likeUb0, and within minutes can increase cytoplasmic Ca2+ concen-tration, as well as diminish membrane cationic current, reduce mito-chondrial membrane potential, and interfere with cell invasion. Wepropose that the benzoquinone ring is responsible for mitochondrialVDAC binding.We conclude that the binding of GA and 17AAG toVDAC inhibits mitochondrial function.

ResultsGA Binds to Mitochondria and VDAC Independent of HSP90. Themotility of MDCK and DBTRG cells is known to be sensitive to

GAdrugs at low concentration (9, 10). Using amodification of theprocedure of Whitesell et al. (11), GA-coupled beads were usedto precipitate potential GA-binding proteins from canine MDCKand human DBTRG cell lysates. As determined by LC-MS/MSanalysis (Table S1), GA-coupled beads precipitate HSP90 asoriginally described by Whitesell et al. (11); in addition, weidentified a 32-kDa protein as the outer membrane mitochondrialvoltage-dependent anion channel protein (VDAC) (Fig. 1A).Although the binding of Hsp90 to GA-beads from cell lysates wasabolished with high concentrations of either GA or 17AAG (Fig.1A, lanes indicated as 17AAG or GA), VDAC binding was notaffected, suggesting different binding mechanisms for GA-HSP90and GA-VDAC.To show that VDAC is readily precipitated frommitochondria,

MDCK and DBTRG cell-derived mitochondria were purifiedfree of HSP90, treated with GA beads, and then precipitated(Fig. 1B). By Western blot analysis, both HSP90 and VDAC wereprecipitated from the postnuclear lysate fraction (lane 1). Themitochondrial pellets were also washed repeatedly to furtherremove the HSP90 until no detectable HSP90 was precipitated(Fig. 1B, lane 2) and only VDAC was detected in the purifiedmitochondrial fraction (Fig. 1B, lane 3). The supernatant fractionfrom each precipitated sample was also collected (Fig. 1B, lanes4–6) to test for remaining HSP90 or VDAC after GA bead pre-cipitation. These results show that VDAC binds directly to GAaffinity beads in the mitochondria and that the binding is in-dependent of HSP90.

Detergent Micelles Interfere with 17AAG–VDAC Binding. VDAC-1 isa highly hydrophobic protein that requires detergent to stabilize itsstructure in vitro by binding with unique hydrophobic sites (12, 13).Different types of detergent can change the secondary structure ofrecombinant VDAC (14), suggesting that the presence of de-tergent (e.g., 1% Triton X-100) in cell lysis buffer may influenceGA-VDAC binding. To test this, we used GA beads to precipitateboth HSP90 and VDAC from total MDCK cell lysates in thepresence or absence of Triton X-100 and with or without free GAto see whether detergent would influence either GA-HSP90 orGA-VDACbinding (Fig. 2A).We found that the addition ofGAat0.1 mM efficiently removes HSP90 from the GA-beads whereas

Author contributions: Q.X., R.W., D.W., H.E.X. and G.F.V.W. designed research; Q.X., R.W.,Y.S., G.C., J.K., R.T., R.B., J.D.-H., E.C., H.O., and D.W. performed research; Q.X., R.W., Y.S.,G.C., Y.X., D.W., H.E.X., and G.F.V.W. analyzed data; and Q.X., R.W., N.R., D.W., andG.F.V.W. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: george.vandewoude@vai.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1015181108/-/DCSupplemental.

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VDAC remains tightly associated. By contrast, VDAC can becompletely dissociated from the GA-beads by detergent in a dose-dependent manner (Fig. 2A). Although 5–10% Triton X-100 issufficient to dissociate the VDAC binding (Fig. 2A,VDAC panel),HSP90-GA binding is not affected (Fig. 2A, HSP90 panel). Sol-utions with 1–2%Triton X-100 and variable concentrations of GA(Fig. 2B) or 17AAG (Fig. 2C) further showed the different bindingcharacteristics of GA and 17AAG to HSP90 and VDAC. Therelease of VDAC is dependent on the concentration of detergentand is consistent with recent findings that detergent micelles de-stabilize VDAC structure (12, 13). Our results suggest that VDACbinding to GA occurs through a hydrophobic association that canbe regulated by detergent micelle formation, especially since GAdrugs are also highly hydrophobic. This is also consistent with thefinding of others (15) that detergent may interfere with measuringthe binding affinities of GA. A spin column assay demonstratinghow detergent micelles influence 17AAG binding is shown in Fig.S1. Although elution buffer without detergent does not interferewith 17AAGbinding toHSP90 (Fig. S1A), detergent micelles highlyinfluence 17AAG binding to purified VDAC (Fig. S1 B and C).

17AAG Binds to Intact Mitochondria. VDAC forms a large voltage-gated pore at the planar bilayer of mitochondria and acts as themajor channel, allowing the flux of both anions and cations, withselectivity varying with the voltage-dependent “open” or “closed”state of the VDAC channel (16). An intact mitochondrial bilayeris essential for VDAC structure and function (17). We thereforeinvestigated the GA–VDAC binding with purified mitochondriausing buffers free of detergent. VDAC was used as a mitochon-drial marker and Hsp90 as a marker for contamination by cytosol(Fig. S2A). Mitochondrial viability was determined by Tetra-methylrhodamine ethyl ester (TMRE) staining followed byFACS to assess metabolic activity (Fig. S2 B and C). We showed

that mitochondria can bind to 3H-17AAG in a dose-dependentfashion, suggesting direct binding between 17AAG and mito-chondria (Fig. S2D).We assessed whether purified full-length HSP90 would com-

pete with mitochondrial VDAC for binding to 3H-17AAG. TheVDAC sources included tumor cell lines, such as glioblastomacells (DBM2, U87), prostate cancer cells (DU145 and PC3), andbreast cancer cells (MDA231 and MCF10), as well as normalorgans such as murine liver, heart, and kidney, and finally MDCKcells. HSP90-MC, a truncated HSP90 lacking the N-terminal GAbinding site, was used as a control (Fig. 3). In all cases, HSP90could effectively compete for 3H-17AAG binding with mitochon-dria regardless of source, whereas HSP90-MC did not (Fig. 3).Taken together, we conclude that GA binding to mitochondriaand VDAC is independent of HSP90 and occurs with mitochon-dria from all sources.

Mitochondrial PTP Inhibition Does Not Correlate with HSP90 Affinity.GA compounds are composed of a benzoquinone moiety attachedto an ansamycin ring (Fig. S3). Ubiquinone (Ub0) is a benzoqui-none molecule known to block the permeability transition pore(PTP) via VDAC (18). Our previous studies showed that hepato-cyte growth factor (HGF) mediated MDCK scattering can beblocked with GA compounds (9, 10). Here we showed that HGF-induced cell scattering was associated with mitochondria relocat-ing to a peri-nuclear position (Fig. 4A), indicating that the in-creased motility is associated with mitochondrial polarization. Totest this, we used a TMRE fluorescence quantification assay andshowed thatHGF induced an increase inmitochondrialmembranepotential in MDCK cells that can be blocked by GA and 17AAGat 0.1 μM (Fig. 4B, P < 0.05). Ub0 was active at ∼80 μM (Fig. 4C,P < 0.05), a concentration also required to inhibit the PTP (19).We questioned whether Ub0 acts as an HSP90 inhibitor andtreatedMDCK cells with GA and 17AAG (Fig. S4). We show thatboth compounds can markedly up-regulate HSP90 and degrade c-Met. Radicicol, a classic HSP90 inhibitor, also followed the sametrend but required log higher concentrations. However, Ub0 didnot affect either the HSP90 or the c-Met level, suggesting that Ub0influences mitochondria directly without affecting HSP90.

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Fig. 1. Geldanamycin-conjugated affinity beads precipitate mitochondrialVDAC independent of HSP90. (A) Control and GA-bead were used to pre-cipitate MDCK and DBTRG cell lysates. Cells were treated with or without17AAG or GA at 1 μM as indicated. (B) GA-coupled bead precipitation ofHSP90 or VDAC from each fraction collected during the mitochondrial pu-rification. Lanes 1–3 are Western blot analyses of the GA affinity beadprecipitates pellet fractions of the postnuclear cell lysate (lane 1), mito-chondrial wash supernatant (lane 2), and solubilized mitochondrial fractions(lane 3). Lanes 4–6 are aliquots of supernatants after GA affinity bead pre-cipitation of the three fractions. All preparations were subjected to immu-noblotting analysis with antibodies to either HSP90 or VDAC.

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Fig. 2. Detergent micelles interfere with VDAC-GA binding but not HSP90-GA binding. Total MDCK cell lysates were used for the GA-bead precipitationand competition assays with various compounds. (A) After GA-bead pre-cipitation, beads were washed and Triton X-100 was added into cell lysisbuffer at different concentrations with or without GA at 0.1 mM to releaseHSP90 and VDAC. (B and C) After GA bead precipitation, beads were washedand resuspended with lysis buffer with Triton X-100 at either 1% or 2%concentration. GA (B) or 17AAG (C) were then added into the samples atdifferent concentration to release HSP90 or VDAC from the beads.

4106 | www.pnas.org/cgi/doi/10.1073/pnas.1015181108 Xie et al.

Geldanamycin and Ub0 inhibit mitochondrial PTP function,but the latter compound does not bind to HSP90. Competitivefilter binding assays with 3H-17AAG yielded a Ki of 0.274 ±0.072 μM for the N-terminal domain of HSP90. This correlatedwith the Kd of 0.79 ± 0.38 μM, which was determined by filterbinding assay for saturation binding and also correlated with thatreported value for the Kd of 17AAG for both the N-terminaldomain of HSP90 and full-length HSP90. In contrast, we foundUb0 and decyl-Ub have no affinity for the N-terminal domain ofHSP90 in filter binding assays at concentrations of 10−4 to 10−11

M. In addition, radicicol, a known inhibitor of HSP90 function,showed strong affinity toward the N-terminal domain of HSP90by competitive binding experiments with a Ki of 21.5 ± 6.8 nM, inline with that previously reported (20–22). These analyses sug-gest that GA compounds, due to the benzoquinone moiety, canbind to mitochondrial VDAC and that benzoquinones influencemitochondria directly.

Intracellular [Ca2+] Efflux Influenced by GA and Ub0. Considering thedecrease of mitochondrial membrane potential by both GA and17AAG,we postulated that drugs with a benzoquinonemoietymayincrease cytoplasmic or internal calcium concentration, [Ca2+]i, bypromoting an efflux from depolarized mitochondria in metaboli-cally compromised cells. We used fluorescence imaging of Fura2-loaded cells to assay the effect of these drugs on [Ca2+]i. Increasingdoses of GA to DBTRG cells increased [Ca2+]i at 10

−8 M, andfurther at 10−7 M (Fig. 5A). This effect of GA was comparablewhether external [Ca2+] ([Ca2+]ext) equaled 1.5 or 0mM (Fig. 5A),indicating that an intracellular source was responsible for the in-creased [Ca2+]i. Thus, we compared effects of the PTP inhibitorUb0 on [Ca2+]i in DBTRG cells. Ub0 (10−5 M) treatment in-creased the magnitude of [Ca2+]i, and the rate of increase wascomparable to that of GA, regardless of the [Ca2+]ext (Fig. 5A),again indicating an intracellular source for the increased [Ca2+]i.We also measured the effect of radicicol, a specific HSP90 in-hibitor lacking the quinone moiety, on [Ca2+]I. Radicicol at either10−7 or 10−5 M had no effect on [Ca2+]i in DBTRG cells (Fig. 5A),ruling out involvement of HSP90 in this effect.

We also tested the influence of GA and Ub0 on MDCK [Ca2+]flux (Fig. 5B). GA increased MDCK [Ca2+]i and DBTRG [Ca2+]iequally. Nevertheless, the baseline [Ca2+]i was ∼10-fold greater inMDCK cells than in DBTRG cells, suggesting very different mem-brane Ca2+ transport properties between the two cell types. Con-sistent with this difference, switching perfusion solution to 0 mM[Ca2+]ext resulted in a rapid diminution of [Ca2+]i, and under thiscondition, GA had no appreciable effect on [Ca2+]i (Fig. 5B).Ub0 increased [Ca2+]i comparably to GA when added to 0 mM[Ca2+]ext; however, it markedly increased [Ca2+]i when [Ca

2+]ext =1.5 mM (Fig. 5B). Radicicol slightly increased [Ca2+]i at 10−5 M(which is 100-fold greater than the effective concentration of GA)when [Ca2+]ext = 1.5 mM but had no effect when [Ca2+]ext =0 mM (Fig. 5B). This indicates that radicicol at 10−5 M stimulatesinflux of external Ca2+ in MDCK cells, as does Ub0 at this con-centration. We have not characterized this CA2+ influx pathwayother than its extracellular source.

GA and Ub0 Inhibit Membrane Cationic Current. The versatility ofCa2+ as a messenger in multiple signaling events requires that theconcentration of calcium ions within the cytoplasm be highly reg-ulated. Release of calcium from the endoplasmic reticulum oftenresults in calcium influx across the cell membrane via store oper-ated calcium entry (SOCE) to replenish the depletion. This influxis most evident by an increase in the Ca2+ release-activated Ca2+

current (ICRAC) of the plasma membrane. To test whether the in-crease in [Ca2+]i by GA and Ub0 occurred by SOCE, we per-formed whole-cell voltage clamp measurements on both MDCKand DBTRG cells to measure the nonselective cationic current.HGF/SF treatment increased this current (Table 1), consistentwith previous findings (23, 24). Dramatically, GA at picomolarlevels inhibited cationic currents within 6 min after addition toeither MDCK or DBTRG cells (Table 1 and Figs. S5 and S6,).Moreover, the VDAC-PTP inhibitors Ub0 and decyl-ubiquinoneboth rapidly decreased membrane cationic current in MDCK cellsat 10 μM (Table 1 and Fig. S5D), a concentration known to inhibitthe PTP (19). We conclude that the benzoquinone moiety of Ub0and of GA are responsible for rapidly (within minutes) reducingmitochondria membrane potential, increasing cytoplasmic/internalcalcium, and inhibitingmembrane cationic current.We propose thatthe inactive or low activity of radicicol in the same assays separatesthe HSP90 chaperone activity from the mitochondria function as-sociated with either cell motility or toxicity.

Blocking Mitochondrial PTP Inhibits Cell Invasion. Calcium releasefrommitochondria can trigger apoptosis and block cell motility (25).Previously we have shown that HGF induces MDCK cell scatteringor glioblastoma cell invasion in parallel with up-regulation of uroki-nase (uPA) activity (9, 10), which can be blocked by GA or 17AAG.Here, we show that GA can diminish HGF-induced membrane cat-ionic current at 10 pM (Table 1). To test whether blocking mito-chondrial PTP pores can inhibit cell motility, we tested the followingknown PTP inhibitors: 4,4′-diisothiocyanatostilbene-2,2′-disulfonicacid, disodium salt (DIDS); 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonic acid, disodium salt (H2DIDS) (26); Ub0; and Decyl-Ub for their ability to inhibitHGF-mediated uPA-plasmin activationas a surrogate for invasion (9) (Fig. 6A) at concentrations that affectmitochondrial function. DIDS is a nonspecific, voltage-dependentVDAC inhibitor preventing O2·

− diffusion from the intermembranespace to the cytoplasm. At 0.5 mM, DIDS inhibits superoxide pro-duction in isolated heart mitochondria (26). Both DIDS andH2DIDS at 100 μM inhibited uPA-plasmin activation. Importantly,the concentrations at which Ub0 and Decyl-Ub significantly inhibitHGF-induced uPA-plasmin activation in MDCK, glioblastomaU251, and DBM2 cells (Fig. 6), are the same drug concentrationsthat block Ca2+ influx in purified mitochondria (18, 19). Again, Ub0displayed the highest activity. We also performed matrigel invasionassay and showed that Ub0 inhibited HGF induced invasion at thesame concentration level as it blocks uPA activity. We conclude that

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Fig. 3. Competitive binding of HSP90 and purified mitochondria with3H-17AAG. Recombinant full-length HSP90 or HSP90 lacking the N-terminal(HSP90MC) were used at different concentrations to compete with purifiedmitochondria isolated from various normal and cancer cells and mouseorgans for 3H-17AAG binding. Mitochondria were isolated from the follow-ing: (A) glioblastoma U87 and DBM2 cells; (B) prostate cancer DU145 and PC-3cells; (C) breast cancer MDA231 and MCF10 cells; and (D) canine epithelialMDCK cells as well as mouse organs, including liver, kidney, and heart.

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mitochondrial PTP inhibitors, includingbezoquinone ansamycin, canblock VDAC functions and prevent cell invasion.

DiscussionHSP90 is the major target of GA and its derivatives, but GAderivatives are also known as antitumor quinones that are both

metabolized by NQO1 (27). Ubiquinone compounds reportedlytarget mitochondria (7, 18), indicating that mitochondria may bean important 17AAG target (4). Our results show that, in a hy-drophobic-dependent manner, 17AAG can physically bind tomitochondrial outer membrane protein VDAC, and the bindingis HSP90 independent (Figs. 1–3).VDAC is a mitochondrial outer membrane protein and is the

key component of the PTP complex, which is responsible formitochondrial metabolite flux (28, 29). VDAC regulation of theopening or closing of the PTP pore is a determinant in mito-chondria-mediated apoptosis and in the generation of redoxpotential (25). We show that VDAC in cell lysates is efficientlypulled down with GA beads, but the specific nature of the hy-drophobic GA-VDAC binding is unknown. Recently, detergentmicelles were shown to be required for the stabilization ofVDAC and for determining VDAC crystal structure. The cal-cium binding site of VDAC has also been mapped to a hydro-phobic region (13). Thus, the VDAC-GA binding may occurthrough hydrophobic regions of VDAC.Others have reported that 0.01% Nonidet P-40 can increase

the binding affinity of HSP90 to GA derivatives 20-fold (15). Ina more recent study, a conserved hydrophobic motif in HSP90 βstrand has been reported to regulate the secretion and functionof HSP90 (30). Thus, the presence of detergent can influence17AAG binding to HSP90, potentially contributing to the poorcorrelation between binding affinity and cytotoxicity (31, 32) orto the potency gap between biochemical binding and Her2 de-gradation (33). We tested detergent by itself and found that17AAG can significantly bind to detergent micelles. This couldbe due to the hydrophobicity of GA derivatives, as a highlywater-soluble molecule such as thymidine is not affected inthese assays (Fig S1). We conclude that GA derivative bindingaffinity can be affected by solvent and detergent; the presence ofdetergent in solution should be below the level of micelle for-mation to provide a more accurate binding affinity or biologicalactivity. Related to this conclusion, our previous study showedthat a subgroup of nine GA compounds and derivatives blockHGF-induced uPA activity at femtomolar concentration with-out degrading c-Met oncoprotein (9, 10, 34). More recently, wefound that the difference was not as large after we adjusted themethod of compound delivery (Table S2). We attribute thisdifference to the hydrophobic properties of nine of the deriva-tives that are affected and in some way correlate with the effi-cacy of inhibiting uPA activity.That GA and benzoquinone drugs increase [Ca2+]i suggests

that they have a common target. It is noteworthy that Ub0 and

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Fig. 4. 17AAG reduces mitochondrial membrane potential. (A) Cell scat-tering and mitochondrial activity were monitored in MDCK cells with JC-1fluorescence. Cells were either “untreated” or “treated” with HGF (100 ng/mL) or further treated with GA (10−8 M). (B and C) Mitochondrial membranepotential was quantified with TMRE uptake assays. HGF enhances TMREuptake in MDCK cells, whereas GA and 17AAG (B), or Ub0 (C) block TMREuptake. *Compared with MDCK control cells without HGF: P < 0.05, Studentt test (n = 4).

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Fig. 5. Effect of increasing dose of benzoqui-none drugs on intracellular Ca2+, [Ca2+]I withDBTRG and MDCK cells. Black indicates externalCa2+ = 1.5 mM; Blue: external Ca2+ = 0 mM.Data are mean ± SE; at least 25 cells weremeasured for each group. (A) Effect of Gelda-namycin, Ub0, and Radicicol on DBTRG [Ca2+]i.(B) Effect of Geldanamycin, Ub0, and Radicicolon MDCK [Ca2+]i.

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Decyl-Ub, known inhibitors of the mitochondrial VDAC andPTP (18), also reduce the plasma membrane cationic currentsimilar to GA. Because both the increase of [Ca2+]i and theinhibition of membrane cationic current occurred rapidly, 6–10min after the addition of GA to the cells (Fig. 5 and Figs. S5 andS6), the mechanism is unlikely to be working through a HSP90client protein affect; rather, we postulate that the mitochondrial

VDAC serves as a target for 17AAG, GA, and Ub0, resulting indepolarization of the mitochondrial membrane potential, a risein [Ca2+]i, and a corresponding decrease in membrane cationiccurrent (i.e., ICRAC) (35, 36). Collapse of the mitochondrialmembrane potential by these drugs disrupts regulation of [Ca2+]ifrom extracellular, ER, and mitochondrial pools. Our resultsalso suggest that the intracellular Ca2+ influx may be a key stepand a potential site for indirectly monitoring the mitochondriaVDAC-PTP response to inhibition by Ub0 and GA. Here, dis-sipation of the mitochondrial membrane potential, by Ub0 andGA, increases [Ca2+]i. Global increases in [Ca2+]i can overrideintracellular Ca2+ gradients and disrupt flickering Ca2+ micro-domains, both of which are necessary to sustain the polarity ofmigrating cells (37).GA derivatives all have a quinone moiety and are metabo-

lized by the NQO1 enzyme (6), indicating that they interact withmitochondria. HSP90 has a broad spectrum of client proteins,and any mitochondrial activity could be masked or considereda subset of HSP90 activity. Previously, studies have suggestedthat GA can regulate ROS generation without affecting HSP90(7). Here we show that quinones, like Ub0, can increase themembrane cationic current (Table 1) and intracellular Ca2+

influx (Fig. 5), as well as block mitochondrial PTP and inhibitcell invasion (Fig.6). This would be expected to add bioactivityto GA compounds, compared to radicicol, without affectingHSP90. The quinone-mediated activity via mitochondrial VDACis important in that VDAC is a major component of mitochon-drial PTP pore, which regulates cell migration, invasion, andapoptosis via Ca2+ regulation and ROS generation (25). It is for

0

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Classic mechanism:

Alterna�ve mechanism:

17-AAG HSP90 degrada�on of downstream onco proteins (eg, c-Met) Block cell invasion

17-AAG Mitochondrial VDAC Intracellular Calcium release

ROS genera�on

Mitochondrial depolariza�on Block cell invasion

D

E Summary of GA mechanism

U251

Fig. 6. Blocking mitochondrial PTP inhibits uPA/invasion. Mitochondrial PTP inhibitors block HGF/SF-mediated uPA activity in MDCK (A), U251 (B), andDBTRG-05MG (C) cells. (D) Ub0 inhibits HGF induced cell invasion in U251 cells at same concentration inhibiting uPA activity. (E) Proposed mechanism of actionof geldanamycin. Free GA either binds to HSP90 via the specific N-terminal ATP binding site or hydrophobically binds to mitochondrial VDAC. Althoughbinding to HSP90 inactivates client protein chaperone activity, resulting in degradation of various oncoproteins, GA binding, through its benzoquinonemoiety, regulates intracellular calcium influx from mitochondria. The increase of intracellular calcium decreases membrane cationic current and reducesmitochondrial metabolism and cell motility (25).

Table 1. Inhibition of HGF/SF-induced cationic currents

Compound concentration (M) Slope conductance* (nS/pF)

Control (no treatment) 0.79 ± 0.10†

Geldanamycin (10−12) 0.31 ± 0.06†,‡

Ubiquinone (10−5) 0.50 ± 0.10†

Decyl-ubiquinone (10−5) 0.64 ± 0.12†

Radicicol (10−11) 0.80 ± 0.28†

HGF/SF (100 scatter units/mL) 1.65 ± 0.19HGF/SF + GA (10−12) 0.47 ± 0.17†

HGF/SF + ubiquinone (10−5) 0.76 ± 0.22†

HGF/SF + decyl-ubiquinone (10−5) 0.36 ± 0.14†

HGF/SF + radicicol (10−11) 1.29 ± 0.52HGF/SF + radicicol (10−8) 1.03 ± 0.03†

MDCK cells were treated with various conditions as indicated. nS, nano-Siemans; pF, picoFarads.*Mean ± SEM.†Differs significantly from HGF/SF treatment, P < 0.05 as determined byStudent Newman-Keuls multiple comparison of means.‡Differs significantly from control, P < 0.05.

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this reason that VDAC has been considered a potential anti-cancer target (38). However, since the ROS generation can alsoinduce cytotoxicity, the safety of targeting VDAC will be an is-sue. Accordingly, the quinone moiety of the GA derivatives isnow being considered for modification to improve both activityand solubility (15). In fact, there are efforts to develop GAderivatives lacking the quinone moiety. Such compounds do notdegrade NQO1 but still show HSP90 inhibition (39), and theymight be more efficacious with reduced toxicity. It is possible thatthe benzoquinone moiety binding to mitochondria may explainthe liver toxicity of 17AAG observed clinically (5, 40, 41). Othershave shown that tumor cells organize an intramitochondrialHSP90 and TRAP-1 chaperone network and regulate mitochon-drial permeability transition (8). However, 17AAG and most GAanalogs do not permeate mitochondria and, thus, require a cell-penetrating linker added to the quinone moiety (8). Consequently,these mitochondrial HSP90 chaperones cannot account for ourfindings on the rapid effects of the unmodified, small-moleculeHSP90 antagonists.Collectively, our results suggest a model in which a subset of

the activity of the GA drugs originates from targeting mito-chondria (Fig. 6E). In this model, the mitochondrial membranepore protein VDAC would be the novel target that binds to GA.

Upon binding, GA, through its benzoquinone moiety, can reg-ulate calcium influx and, in turn, decrease the cell membranecationic current, inhibiting cell invasion and synergizing with theHSP90 effect to cause an anti-cancer effect on motility (Fig. 6D).However, targeting mitochondria could also account for toxicity,e.g., through ROS generation. Because this activity is in-dependent of inhibiting HSP90 chaperone function, diminishingthe benzoquinone moiety’s toxicity in future drugs should beconsidered (39, 40).

Materials and MethodsSI Materials and Methods provides additional information related to themain text on the following topics: cell lines and drugs, GA-immobilizedaffinity beads and GA bead precipitation assays, release of HSP90 and VDACfrom GA-conjugated beads with free GA, mass spectrometry analysis,HSP90, and HSP90MC purification, 3H-17AAG binds to purified mitochon-dria, competitive binding to 3H-17AAG between purified mitochondria andHSP90, TMRE measurement of mitochondria membrane potential, whole-cell voltage clamp, Ca2+ measurements by fluorescence imaging of Fura2,confocal microscopy, HGF/SF-Met-uPA-plasmin assay, and Matrigel inva-sion assay.

ACKNOWLEDGMENTS. We thank David Nadziejka and Julia A. Patzelt forediting the manuscript.

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