Dehydro-α-lapachone, a plant product withantivascular activityIgor Garkavtseva,1, Vikash P. Chauhana,b, Hon Kit Wonga, Arpita Mukhopadhyayc, Marcie A. Glicksmand,Randall T. Petersonc, and Rakesh K. Jaina

aEdwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; bHarvardSchool of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138; cCardiovascular Research Center, Massachusetts General Hospital,Charlestown, MA 02129; and dPartners Center for Drug Discovery, Brigham and Women’s Hospital and Harvard Medical School, Cambridge, MA 02139

Edited* by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved May 13, 2011 (received for review March 15, 2011)

Antivascular agents have become a standard of treatment formany malignancies. However, most of them target the VEGFpathway and lead to refractoriness. To improve the diversity ofoptions for antivascular therapy, we applied a high-throughputscreen for small molecules targeting cell adhesion. We thenassayed the resulting antiadhesion hits in a transgenic zebrafishline with endothelial expression of EGFP (Tg(fli1:EGFP)y1) to iden-tify nontoxic molecules with antivascular activity selective to neo-vasculature. This screen identified dehydro-α-lapachone (DAL),a natural plant product. We found that DAL inhibits vessel regen-eration, interferes with vessel anastomosis, and limits plexus for-mation in zebrafish. Furthermore, DAL induces vascular pruningand growth delay in orthotopic mammary tumors in mice. Weshow that DAL targets cell adhesion by promoting ubiquitinationof the Rho-GTPase Rac1, which is frequently up-regulated in manydifferent cancers.

drug discovery | angiogenesis

Regulation of blood vessel growth has proven to be an im-portant strategy in the treatment of cancer and may provide

new targets for the treatment of inflammatory disorders, asthma,obesity, diabetes, multiple sclerosis, endometriosis, and bacterialinfections (1, 2). Antiangiogenic agents, most of which targetVEGF or its receptors, are emerging as standard therapies forseveral major human cancers (3–5). Unfortunately, antiangio-genic therapy leads to modest efficacy, inherent or acquired re-sistance, and rare but life-threatening toxicity (6, 7). Consideringthese limitations, the development of antivascular agents thattarget pathways other than VEGF is urgently needed.Cell adhesion pathways present attractive targets for anti-

vascular agents in cancer therapy. Endothelial cell (EC) adhe-sion is crucial for blood vessel function (8, 9). Blood vessels intumors are abnormal, featuring unusual leakiness, high tortuos-ity, and inefficient network structure (10). The immature ECs ofthis abnormal neovasculature in tumors have weak cell-celljunctions (10–13)—likely making them particularly sensitive toantiadhesion therapies. Candidate antiadhesion agents for anti-vascular therapy have been identified and have shown promisingresults in preclinical studies (14). Unfortunately, these early at-tempts at development of antiadhesion agents for antivasculartherapy were met with failure stemming from issues with toxicity.We hypothesized that a nontoxic compound targeting cell adhesionwould produce a safe antivascular effect specific to neovasculature.To this end, we devised a high-throughput cell adhesion screen toidentify candidate nontoxic antiadhesion compounds with anti-vascular activity in tumors.

Results and DiscussionScreening Yields a Natural Product—DAL. We developed a uniquescreening strategy to identify potential agents that target adhe-sion of ECs or cancer cells to their substrate. To this end, webegan by screening 50,000 compounds in a high-throughputmanner (Fig. 1A). Our initial screening step quantified the

number of cells remaining attached to their wells after in-cubation with each compound and subsequent washing steps.Only 86 compounds affected cellular adhesion in our assay. Celladhesion adaptor proteins have a domain for binding actin fila-ments (15, 16), thus adhesion molecules are directly linked to theactin cytoskeleton. We reasoned that the agents that affect celladhesion may be monitored through remodeling of actin fila-ments. As our second screening step, we explored the effect ofthe selected compounds on actin assembly and redistribution byfixing and staining-treated cells with phalloidin. We observedchanges in actin assembly and cell shape after treatment with 12compounds. We next prioritized this set for compounds thatwere nontoxic to normal cells, cancer cells, and zebrafish. Ofthese 12 compounds, two were selected based on the in vitroand in vivo assessment. One of these compounds, dehydro-α-lapachone (DAL; Fig. 1B), showed structural similarities toβ-lapachone, which has demonstrated antitumor and anti-trypanosomal activities through DNA topoisomerase I in-hibition and prevention of DNA repair (17, 18). We furthertested DAL for toxicity in SCID mice, and found that mice aretolerant to i.p. doses of up to 100 mg/kg with no signs of tox-icity. We therefore selected DAL—a natural product from theTabebuia Avellaneda tree—for further study.

DAL Shows Antivascular Effects in Zebrafish Models. To elucidatethe potential impact of DAL on the process of vascular networkformation, we next assessed its effects in zebrafish embryos atdifferent stages of development. To visualize vessel defects indetail, we used transgenic fish expressing EGFP in endothelialcells (Tg(fli1:EGFP)y1) (19, 20). This model expresses EGFP inblood vessel ECs throughout normal development and during finregeneration (21). We first characterized the effects of DAL onnormal vascular development in zebrafish embryos. In controlembryos, developing vessels migrated from the lateral platemesoderm to the midline, where they coalesced into a vascularcord. These endothelial clusters subsequently established thepattern of the dorsal aorta and posterior cardinal vein. Inter-somitic vessels sprouted at designated branch sites in controlembryos after dorsal aorta formation. In contrast, after treat-ment with DAL, the sprouting of intersomitic vessels at theirdesignated branch sites failed to occur and the embryos de-veloped unusual wave-like vessel structures (Fig. 2A). DAL

Author contributions: I.G., V.P.C., H.K.W., A.M., M.A.G., R.T.P., and R.K.J. designed re-search; I.G., V.P.C., H.K.W., A.M., M.A.G., and R.T.P. performed research; V.P.C. contrib-uted new reagents/analytic tools; I.G., V.P.C., H.K.W., A.M., M.A.G., and R.T.P. analyzeddata; I.G., V.P.C., H.K.W., and R.K.J. wrote the paper.

Conflict of interest statement: R.K.J. received commercial research grants from Dyax,AstraZeneca, MedImmune and Roche; consultant fees from AstraZeneca, Dyax, Astellas,SynDevRx, Regeneron, Genzyme, Morphosys, and Noxxon Pharma; and a speaker hono-rarium from MPM Capital. R.K.J. owns stock in SynDevRx. No reagentsor funding fromthese companies was used in these studies. There is no significant financial or othercompeting interest in the work.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: igorg@steele.mgh.harvard.edu.

11596–11601 | PNAS | July 12, 2011 | vol. 108 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1104225108

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0

treatment resulted in a profound reduction in the complexity ofthe arterial network. These findings demonstrate that DAL caninterfere with the regulation of vascular formation and branchingmorphogenesis during development. Interestingly, DAL did notshow any effects on the vasculature in adult fish, probably dueto the stable junctions and postmitotic nature of the maturevasculature.To examine whether DAL has a similar antivascular effect on

neovasculature in adult zebrafish, we induced neovascularizationby amputating the caudal fin at the ≈50% proximal–distal leveland imaged fin rays and vessels from each fish over time. Incontrol zebrafish caudal fins, amputated blood vessels healedtheir ends by 24 h after amputation and then reconnectedarteries and veins through anastomosis, with blood flow resumingat wound sites by 48 h after amputation (Fig. 2B). Meanwhile,regenerating vessels in fish treated with DAL had defects inanastomosis and plexus formation. All control fish developednormal blood vessels and formed anastomotic bridges at theamputation plane by 9 d after amputation; in contrast, we didnot find such bridges in fish treated with DAL (Fig. 2C).

DAL Prunes Tumor Vasculature. Because the zebrafish data in-dicated that DAL is a potential antivascular agent selective forneovasculature, we sought to determine the effects of DAL ontumor vasculature in mammals. To study these effects quanti-tatively, we conducted fluorescent angiographies in female SCIDmice bearing orthotopic 4T1 mammary tumors in mammary fatpad windows via intravital multiphoton microscopy (Fig. 3A)(22–24). We treated these mice with 37.5 mg/kg DAL or salinedaily for 5 d by i.p. injection. We found that DAL treatmentdecreases tumor vascular volume fractions—a measure of vas-cular density—compared with saline treatment (Fig. 3B; P =0.002, day 4). Furthermore, DAL treatment lowers total tumorvascular length (normalized to tumor volume) versus salinetreatment (Fig. 3C; P = 0.007, day 2; P = 0.02, day 4), whereasmean tumor vascular diameter remains the same (Fig. 3D).

These data indicate that DAL reduces vascular density in tumorsthrough vessel pruning.

DAL Inhibits Tumor Growth. Considering that DAL has an anti-vascular effect in orthotopic mammary tumors in mice, weexpected that it might have an antitumor effect. Pharmacokineticstudies for DAL indicated that its half-life is 1.7 h in plasma afteri.p. dosing. We evaluated the effect of DAL on the growth oforthotopic 4T1 and E0771 mammary tumors with daily treat-ment of DAL or saline (Fig. 4A). We used 37.5 mg/kg DAL forthese experiments based on the pharmacokinetics results as wellas in vitro and in vivo data. In E0771 tumors, with treatmentinitiated at a ≈100 mm3 volume, DAL increased the tumorvolume doubling time from 2.65 ± 0.27 d to 4.77 ± 0.58 d (P =0.01). In 4T1 tumors, with treatment starting at a larger tumorvolume of ≈200 mm3, DAL increased the doubling time from2.20 ± 0.07 d to 11.21 ± 3.53 d (P = 0.04; Fig. 4B). Importantly,DAL induced no weight loss or other noted signs of toxicity inthese mice. Together, these results demonstrate that DAL is asafe chemical agent with prominent antitumor effect.

DAL Interferes with Adhesive Properties of Endothelial Cells. To gainfurther insight into the antivascular effects of DAL and to testthe involvement of DAL in modulating actin assembly, we platednormal human umbilical vein endothelial cells (HUVEC) andtreated them with 10 μM DAL for 3 h. We fixed, permeabilized,and subsequently stained the cells with phalloidin to visualizeF-actin (Fig. 5A). Cells treated with DAL were smaller thanuntreated control cells and had a rounded shape. Actin stressfibers were clearly visible and aligned with the long axis in con-trol cells, whereas DAL-treated cells featured actin present onlyas small fibers and spots throughout the cells or accumulatedclose to the nuclei. Together, these results show that DALdestroys or significantly changes the normal organization of theactin cytoskeleton and further inhibits actin-dependent processessuch as cell spreading on the extracellular matrix.

O

O

CHCH

A B

D

E

CO

Fig. 1. A high-throughput drug screen for the DAL. (A) Of the 50,000 compounds screened, we selected 86 to test in the secondary assays. We only selectedcompounds that made MDA-MB-231 cells more rounded, with less spreading than control cells, and interfered with actin regulation for further study. Wefurther passed all these selected small molecules through a number of filtering toxicity assays to eliminate toxic compounds. To confirm the absence oftoxicity in the cellular model for each candidate molecule, we repeated the experiments and reassayed at three different concentrations (1.67 M, 5 M, and10 M). We then analyzed the nontoxic compounds in a zebrafish model. We used small molecules that did not alter zebrafish survival during embryonicdevelopment and adult life for further experiments. Finally, of 50,000 compounds, we selected two nontoxic compounds that demonstrated antivascularproperties. One of these compounds, DAL, was chosen for further experiments. (B) The structure of DAL, a natural product. (C) DAL-treated cells lost theiradhesion properties. (D) Untreated MDA-MB-231 control cells spread more rapidly on fibronectin-coated plates than cells treated with DAL. In contrast, cellstreated with DAL were significantly smaller than control cells and had a rounded shape. (E) DAL is involved in actin cytoskeleton assembly regulation. ForF-actin visualization, we plated MDA-MB-231 cells on fibronectin and treated them with each compound (5 μM) for 2 h. After fixing and permeabilizing thesecultured cells, we subsequently stained them with phalloidin. The untreated control cells spread more rapidly on fibronectin-coated plates than those treatedwith DAL. In contrast, cells treated with DAL were significantly smaller than control cells and had a rounded shape. In the control cells, actin stress fibers wereclearly visible and aligned with the long axis of the cells. Meanwhile, in cells treated with DAL, actin was accumulated close to the membrane. We usedcytochalasin D as a positive control that inhibits actin assembly.

Garkavtsev et al. PNAS | July 12, 2011 | vol. 108 | no. 28 | 11597

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0

To test whether DAL affects EC functions, we used in vitromodels of vascular network formation from HUVECs. Afterovernight cultivation in matrigel, HUVECs spontaneously as-semble into cord-like structures that resemble vascular networks.Treatment with DAL not only blocks HUVEC network forma-tion, but also leads to complete reorganization of existing net-works over a few hours (Fig. 5B). These results indicate that DALlikely interferes with cell-cell junction integrity and actin assemblyin ECs as a mechanism for its antivascular effects. Because DALaffects actin cytoskeleton assembly, we speculated that DALmight also affect cell motility. We used a scratch wound assay tostudy HUVEC motility based on the healing rate of a woundedmonolayer. Cells treated with DAL did not move and failed toclose the wound (Fig. 5C). These results demonstrate that DALcan interfere with EC motility as well, likely representing a sec-ondary mechanism for inhibiting angiogenic sprouting.

DAL Decreases Rac1 Activity and Promotes Degradation. Consideringthat DAL brings about its antivascular effects by destabilizing celladhesion through the actin cytoskeleton, we sought to identifywhich specific adhesion pathway it targets. Mechanical attach-

ment at cell-cell adherens and tight junctions is regulated by dy-namic changes in the actin cytoskeleton. Adherens junctions arecomposed of E-cadherin/β-catenin complexes and are connectedto the actin cytoskeleton via α-catenin (25, 26), whereas tightjunctions consist of transmembrane claudins and occludin and areassociated with the actin cytoskeleton via ZO proteins (27, 28).We therefore assayed for potential targets of DAL in signalingpathways involved in actin remodeling. We found that DAL hadno significant effects on 14 kinases (AKT1, AKT2, AKT3, PKA,PKCα, PKCδ, PAK1, EPHB2, EPHB4, p38α, PI3K-α, PI3K-β,PI3K-γ, PI3K-δ). We then suspected that Rho-GTPase familymembers might be targets for DAL. In particular, we reasonedthat Rac1 might be the target because of the combination ofdisrupted adhesion and actin networks along with inhibited mo-tility. Rac1 plays an important role in actin cytoskeletal organi-zation and is involved in the coordination of cell adhesion (29).Furthermore, Rac1 is responsible for stabilizing endothelial celljunctions by opposing actin remodeling by Rho (30). To testwhether DAL might modulate Rac1, RhoA, and Cdc42 activities,we used affinity purification assays to monitor RhoA and Cdc42/Rac1 activity. We applied the Rho-binding domain (RBD) of theRho effector protein rhotekin and the p21 binding domain (PBD)of the Cdc42/Rac effector protein p21 activated kinase 1 (PAK)to pull down active forms of each protein. We found that DALdecreased Rac1 activity in a concentration-dependent manner(Fig. 6A)—and seemed to decrease Rac1 levels—but had no ef-fect on Cdc42 or RhoA activity, indicating Rac1 regulators orRac1 itself as potential targets of DAL.Treatment of cells with DAL did not change expression levels

of Rac1 mRNA measured with quantitative PCR. We confirmedthat DAL reduces protein levels of Rac1 (Fig. 6B), and wetherefore suspected that DAL promotes Rac1 protein degrada-

A control DAL

B control DAL

C control DAL

Fig. 2. DAL impairs vessel development and regeneration in zebrafish. (A)Macroscopic view of 48 h after fertilization Tg(fli1:EGFP)y1 zebrafish em-bryos, comparing control embryos and embryos treated with DAL (5 μM).Treated embryos failed to form vessel branches. (B and C) Regenerationexperiments with zebrafish, depicting the vasculature of the caudal fin on 2and 9 d after amputation in control and treated zebrafish. White lines in-dicate the amputation plane. (B) Vascular plexus formation, plexus remod-eling, and late regenerative angiogenesis. (C) A higher magnification of thereconnected vessels. Nine days after amputation, the regenerating vascula-ture of each fin ray (three fin rays are shown) consists of a plexus with denseunstructured vessels extending distally from the amputation plane (whitearrowheads). Regenerating blood vessels in the wild-type control (fli1:EGFP)form plexuses. Fish treated with DAL have defects in anastomosis and plexusformation. (Scale bars: 200 μm.)

depth ( m)

A B

C

D

Fig. 3. DAL has antivascular effects in mouse models. (A) Representativeintravital images of tumor vessels in mice bearing orthotopic 4T1 mammarytumors through a treatment course. We imaged the tumors with multi-photon microscopy on days 0, 2, and 4; daily treatments started on day0 after the first images were collected. Images are 3D projections of tissuefrom 0 to 200 μm, with pseudocolor labeling by depth. (B–D) Measuredvascular parameters in the mice over the course of treatment. Tumors inmice treated with DAL have lower vascular volume fractions than tumors insaline-treated mice at day 4 (P = 0.0017). Total tumor vascular length (nor-malized to tumor volume), but not mean tumor vascular diameter, is lowerin DAL-treated mice than in saline-treated mice (P = 0.0069, day 2; P = 0.024,day 4), suggesting vessel pruning as the mechanism for decreased vascularvolume fraction. n = 5 for the saline group, n = 4 for the DAL group.

11598 | www.pnas.org/cgi/doi/10.1073/pnas.1104225108 Garkavtsev et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0

tion. We questioned whether DAL might enhance Rac1 ubiq-uitination and therefore speed up degradation (31–34). For thedetection of Rac1 ubiquitination in HUVECs, we identifiedRac1-ubiquitin binding in situ by using a Duolink molecularproximity assay. In this assay, molecules thought to interact aretargeted with fluorescent antibodies in situ, and individual inter-actions generate a signal detectable by microscopy only if sec-ondary antibodies are located in close proximity to each other.For detection of ubiqutinated Rac1 proteins, we used a mouseantibody raised against Rac1 protein and a rabbit antibody raisedagainst a ubiquitin. We used low concentrations of irreversible(lactacystin) and reversible (MG-132) proteasome inhibitors toblock the ubiquitin proteasome system and enrich the fraction ofubiquitinated proteins in our assay. Cells treated with DAL con-tained more ubiquitinated Rac1 protein compared with controlcells (Fig. 6C). These results strongly suggest that DAL promotesRac1 ubiquitination, leading to rapid Rac1 degradation.

ConclusionAngiogenesis inhibitors targeting the VEGF signaling pathwayhave proven to be an efficacious anticancer treatment strategy.Novel targets for drug discovery are needed to diversify anti-angiogenic treatment and enhance safety for the benefit ofcancer patients. To improve the diversity of options for anti-vascular therapy, we applied a high-throughput screen for smallmolecules that affect cell adhesion. We identified a nontoxicnatural plant product, DAL, with antivascular properties leadingto an antitumor effect. Intriguingly, DAL affects cell adhesion bypromoting the ubiquitination and proteasomal degradation ofthe Rho-GTPase Rac1, which has been noted to play a role incell-cell adhesion stability. This small molecule may serve asa lead compound for the development of a novel class of anti-cancer drugs.

Materials and MethodsCompound Library.We used a compound library from the Partners Center forDrug Discovery (PCDD) high-throughput assay. This library includes 50,000compounds from different sources: (i) marketed drugs from Prestwick; (ii)purified natural products; (iii) 3,000 end-blocked tetrapeptides; (iv) smallmolecules purchased from Peakdale, Maybridge, CEREP, Bionet ResearchLtd., Chemical Diversity Lab, ENAMINE, and I.F.Lab Ltd; and (v) small mole-cules from PCDD chemists and from different academic institutions. Com-pounds are stored at a stock concentration of 2 mg/mL to ≈5 mM. For thescreen, the 5 mM solutions were diluted to ≈1.67 mM in 100% DMSO, and0.4 μL of each was spotted in wells in 384-well plates by using a BeckmanCoulter Multimek 96/384 Channel Automated Pipetter. The average molec-ular weight of the compounds in the library is 400 Da (range = 225–600), andthis library has been tested successfully in a number of screening assays.

High-Throughput Screening (HTS). We used a cell-based readout system forcompound selection. For our cell-based screening, we selected an optimal celldensity of 10,000 breast cancer MDA-MB-231 cells per well to produce the

most prominent signal. We incubated the plated cells with each compound,washed with PBS, and used the Cell Titer-Glo (Promega) reagent to detect thenumber of cells remaining attached in each well. This ATP-based detectionhas a linear relationship between the luminescent signal and the number ofcells per well. Of the 50,000 compounds screened, we selected 86 (0.172% ofscreened compounds) at 1.67 μM concentration for the second screeningstep. We treated breast cancer cells with the compounds at 3 μM for 3 h andthen examined for changes in cell shape by microscopy. We selected 12 ofthe 86 compounds for further study. We passed all these selected com-pounds through a number of toxicity assays to eliminate any toxic com-pounds. We confirmed the absence of toxicity for the candidate compoundsin the cellular model in repetitive experiments and reassayed at three dif-ferent concentrations (1.67, 5, and 10 μM). We analyzed the compounds thatshowed no toxicity at any concentrations tested in a zebrafish model and

0

4

8

12

16

4T1 E0771

salineDAL

*

**

B

tum

or s

ize

(mm

₃ )

A

0

400

800

1200

0 4 8 12 16 20

4T1 salineDAL

0

400

800

1200

0 4 8 12 16 20

E0771

Fig. 4. DAL has antitumor effects in orthotopic mouse models. (A) Tumor growth curves for 4T1 (n = 4) and E0771 orthotopic mammary tumors (n = 5). Wetreated tumor-bearing female SCID mice with daily i.p. injections of saline or 37.5 mg/kg DAL. Throughout the study, we measured tumor size on alternatingdays. Because all tumors grew at different rates to the start size, we time- and size-matched tumors for their initial treatment on day 0 just after the firstmeasurement. (B) DAL increased the doubling time for 4T1 tumors from 2.20 ± 0.07 d to 11.21 ± 3.53 d (P = 0.044) and that for E0771 tumors from 2.65 ± 0.27 d to4.77 ± 0.58 d (P = 0.010).

B control DAL

A control DAL

C control DAL

Fig. 5. DAL interferes with adhesive properties of endothelial cells in vitro.(A) DAL is involved in actin cytoskeleton assembly regulation. For F-actinvisualization, we plated HUVECs and treated them with saline or DAL (10μM) for 3 h. After fixing and permeabilizing these cultured cells, we sub-sequently stained them with phalloidin. (B) DAL interferes with adherensjunctions in networks of HUVEC cells cultured on matrigel. After 24 h, con-trol HUVEC cells organized into a network of cordlike structures, whereasDAL-treated HUVECs show disjoined networks. Existing cordlike structures,when exposed to 10 μM of DAL for 3 h, were also disrupted. (C) A wound-healing motility assay with HUVECs. We disrupted HUVEC monolayersmechanically with a pipette tip and followed the migration of cells to “heal”the wound using microscopy. DAL (5 μM) slowed healing compared with thecontrol, shown at 24 h after wounding.

Garkavtsev et al. PNAS | July 12, 2011 | vol. 108 | no. 28 | 11599

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0

then used compounds at concentrations that did not affect zebrafish sur-vival during embryonic development and adult life for further experiments.Of the 50,000 compounds, we selected the compound DAL for furthercharacterization. As a further test of toxicity, we injected DAL i.p. at 100 mg/kg in SCID mice and monitored for signs of toxicity including cachexia, ruf-fled fur, diarrhea, anorexia, skin ulceration, and toxic death.

Zebrafish Maintenance and Drug Eexposure.We raised zebrafish in accordancewith established protocols (19, 20). The age of embryos is indicated by thehours after fertilization and days after fertilization for all experimental datashown. For the screening study, we incubated zebrafish embryos with theselected compounds at a concentration of 5–10 μM. We then examinedtreated embryos under a 100× PlanNeofluor objective mounted on a NikonTE-200 epifluorescence microscope. Images of embryos bearing GFP-positivecells were captured with a Zeiss stereomicroscope.

Regeneration Experiments. For the regeneration experiments, we ampu-tated caudal fins at approximately the 50% proximal-distal level (21). Wekept the amputated fish in individual 250-mL fish tanks and used at leastfive fish in each group. We carried out staging for normal blood vesselregeneration at 25 °C with TG(fli1:EGFP)y1 fish, with the same fin ray andvessels of each fish photographed at different time intervals during thecourse of the time-lapse study.

Determination of Rac1 Activity. We measured Rac1 and cdc42 activities witha Rac1 (BK035) activation assay biochem kit (Cytoskeleton) according to themanufacturer protocol. In brief, we bovine brain extract-starved HUVECs for12 h in 1% FBS-EGM (Lonza) and pretreated with various concentrations of

DAL for 30 min at 37 °C. We then stimulated the HUVECs with VEGF at 50 ng/μl for 15 min at 37 °C. After stimulation, we immediately incubated the cellson ice, washed twice with PBS, and lysed with lysis buffer. We precleared thelysates and added 15 μL of PAK-PBD beads for pull-down of activated Rac1or cdc42. After rocking at 4 °C for 1 h, we washed the beads once and boiledthem at 100 °C for 2 min. We then resolved the protein lysates in a 8–16%gradient gel (Invitrogen) and transferred them to a PVDF membrane. Weadded primary antibodies (anti-Rac1, 1:1,000, cytoskeleton or anti-cdc42,1:1,000, Cell Signaling) to bind to the protein overnight at 4 °C, followed byincubation with HRP-conjugated secondary antibodies. We measured pro-tein activity by the standard ECL method (Amersham).

Measurement of Vascular Parameters in Tumors in Mice. We treated femaleSCID mice bearing orthotopic GFP-labeled 3-mm 4T1 mammary tumors withfive daily i.p. injections (day 0–day 4) of 37.5 mg/kg DAL or saline. We sur-gically implanted mammary fat pad windows (35) to facilitate intravitalimaging through multiphoton microscopy with a custom-built multiphotonmicroscope with a 20× 0.95 N.A. objective (Olympus) and imaged mice byfluorescent angiography after retro-orbital injection of 60 μL of 10 mg/mLTAMRA-dextran (2 MDa molecular mass). We collected images on days 0, 2,and 4, with the first images collected immediately before the first treat-ment. We imaged 2 × 2 grids in the sample plane (630-μm field of view ateach position) tissue from 0 to 200 μm depth. To measure vascular param-eters in the mice over the course of treatment, we applied a custom 3Dvascular tracing and analysis software package based on MATLAB (Math-works) to the images.

Tumor Growth Delay Study. To assess the antitumor effects of DAL, we treatedfemale SCID mice bearing orthotopic 4T1 or E0771 mammary tumors withdaily i.p. injections of 37.5 mg/kg DAL or saline. To calculate tumor volumes,wemeasured themajor andminor axis diameters with calipers and calculatedthe tumor volume assuming a spherical shape based on the average of thesetwo dimensions. We implanted the tumors in the mammary fat pad andallowed them to grow to a starting size (small ≈100 mm3 tumors for E0771,large ≈200 mm3 tumors for 4T1) over ≈2 wk. To ensure that tumors in thetwo groups were of similar growth rates, we began the treatment withsaline or DAL in pairs of tumors with similar sizes and growth times. Wemeasured tumor volumes every other day until the tumors reached andaverage of ≈10 mm in diameter. We quantified tumor growth with thedoubling rate, calculated by fitting an exponential growth curve to the datafor tumor size versus time.

Kinase Inhibition Screening. To determine whether DAL might target kinaseactivity, we screened against kinases (AKT1, AKT2, AKT3, PKA, PKC-α, PKC-δ,PAK1, EPHB2, EPHB4, p38-α, PI3K-α, PI3K-β, PI3K-γ, PI3K-δ) in a 10-dose IC50mode with threefold serial dilutions starting at 100 μM. We used staur-osporine as a control starting at 10 μM and carried out the reactions at1 μM ATP.

ACKNOWLEDGMENTS. We thank Brian Seed for his insightful input through-out this project, various members of the Steele Laboratory—Gang Cheng,Sergey Kozin, and Ned Kirkpatrick—for help with experiments, and LanceMunn and Dai Fukumura for the helpful comments and suggestions. Thiswork was supported in part by Department of Defense Breast Cancer Re-search Innovator Award W81XWH-10-1-0016 (to R.K.J.), National Institutesof Health Grant HL079267 (to R.T.P.), and a grant from Partners (to I.G.).

1. Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of

angiogenesis. Nature 473:298–307.2. Folkman J (2007) Angiogenesis: An organizing principle for drug discovery? Nat Rev

Drug Discov 6:273–286.3. Chung AS, Lee J, Ferrara N (2010) Targeting the tumour vasculature: Insights from

physiological angiogenesis. Nat Rev Cancer 10:505–514.4. Nagy JA, Chang SH, Dvorak AM, Dvorak HF (2009) Why are tumour blood vessels

abnormal and why is it important to know? Br J Cancer 100:865–869.5. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L (2006) VEGF receptor signalling -

in control of vascular function. Nat Rev Mol Cell Biol 7:359–371.6. Bergers G, Hanahan D (2008) Modes of resistance to anti-angiogenic therapy. Nat Rev

Cancer 8:592–603.7. Jain RK, et al. (2009) Biomarkers of response and resistance to antiangiogenic

therapy. Nat Rev Clin Oncol 6:327–338.8. Carmeliet P, et al. (1999) Targeted deficiency or cytosolic truncation of the VE-

cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.

Cell 98:147–157.

9. Corada M, et al. (1999) Vascular endothelial-cadherin is an important determinant of

microvascular integrity in vivo. Proc Natl Acad Sci USA 96:9815–9820.10. Jain RK (2005) Normalization of tumor vasculature: An emerging concept in

antiangiogenic therapy. Science 307:58–62.11. Dejana E (2004) Endothelial cell-cell junctions: Happy together. Nat Rev Mol Cell Biol

5:261–270.12. Hashizume H, et al. (2000) Openings between defective endothelial cells explain

tumor vessel leakiness. Am J Pathol 156:1363–1380.13. Hobbs SK, et al. (1998) Regulation of transport pathways in tumor vessels: Role of

tumor type and microenvironment. Proc Natl Acad Sci USA 95:4607–4612.14. Corada M, et al. (2002) A monoclonal antibody to vascular endothelial-cadherin

inhibits tumor angiogenesis without side effects on endothelial permeability. Blood

100:905–911.15. Gumbiner BM (2005) Regulation of cadherin-mediated adhesion in morphogenesis.

Nat Rev Mol Cell Biol 6:622–634.16. Borisy GG, Svitkina TM (2000) Actin machinery: Pushing the envelope. Curr Opin Cell

Biol 12:104–112.

A B

C

Fig. 6. DAL decreases Rac1 activity and activates Rac1 degradation. (A)Detection of RhoA activity in HUVEC cells treated with DAL. (B) DALdecreases level of Rac1 in HUVECs. We treated HUVEC cells with 10 μM DALfor 24 h and were lysed. A Western blot with anti-human Rac1 antibody isshown (Upper), whereas Lower represents a loading control. (C) Treatmentwith DAL increases Rac1 ubiquitination in HUVEC cells. To enhance ubiqu-tination events, we pretreated HUVEC cells with the proteasome inhibitorslactacystin (2.5 mM) or MG-132 (0.2 mM) for 1 h and subsequently treatedcells with DAL (10 μM) for 24 h. We used Duolink in situ for the detection ofubiqutinated Rac1 in DAL-treated and -untreated HUVEC cells. For the twoDuolink PLA probes, we used primary mouse antibodies raised against hu-man Rac1 and primary rabbit anti-ubiquitin. Fluorescent spots representubiqutinated Rac1. We additionally stained the nuclei with DAPI.

11600 | www.pnas.org/cgi/doi/10.1073/pnas.1104225108 Garkavtsev et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0

17. Li CJ, Averboukh L, Pardee AB (1993) beta-Lapachone, a novel DNA topoisomerase Iinhibitor with a mode of action different from camptothecin. J Biol Chem 268:22463–22468.

18. Planchon SM, et al. (1995) Beta-lapachone-mediated apoptosis in human promyelocyticleukemia (HL-60) and human prostate cancer cells: A p53-independent response.Cancer Res 55:3706–3711.

19. Huang CC, Lawson ND, Weinstein BM, Johnson SL (2003) reg6 is required forbranching morphogenesis during blood vessel regeneration in zebrafish caudal fins.Dev Biol 264:263–274.

20. Whitehead GG, Makino S, Lien CL, Keating MT (2005) fgf20 is essential for initiatingzebrafish fin regeneration. Science 310:1957–1960.

21. Nechiporuk A, Keating MT (2002) A proliferation gradient between proximal and msxb-expressing distal blastema directs zebrafish fin regeneration. Development 129:2607–2617.

22. Brown E, et al. (2003) Dynamic imaging of collagen and its modulation in tumorsin vivo using second-harmonic generation. Nat Med 9:796–800.

23. Garkavtsev I, et al. (2004) The candidate tumour suppressor protein ING4 regulatesbrain tumour growth and angiogenesis. Nature 428:328–332.

24. Vakoc BJ, et al. (2009) Three-dimensional microscopy of the tumor microenvironmentin vivo using optical frequency domain imaging. Nat Med 15:1219–1223.

25. Anderson JM, Van Itallie CM (2008) Tight junctions. Curr Biol 18:R941–R943.26. Miyoshi J, Takai Y (2005) Molecular perspective on tight-junction assembly and

epithelial polarity. Adv Drug Deliv Rev 57:815–855.

27. Tzima E (2006) Role of small GTPases in endothelial cytoskeletal dynamics and the

shear stress response. Circ Res 98:176–185.28. Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev

Mol Cell Biol 2:285–293.29. Ridley AJ (2006) Rho GTPases and actin dynamics in membrane protrusions and vesicle

trafficking. Trends Cell Biol 16:522–529.30. Wojciak-Stothard B, Ridley AJ (2002) Rho GTPases and the regulation of endothelial

permeability. Vascul Pharmacol 39:187–199.31. Reinstein E, Ciechanover A (2006) Narrative review: Protein degradation and human

diseases: The ubiquitin connection. Ann Intern Med 145:676–684.32. Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome

system by protein aggregation. Science 292:1552–1555.33. Wong HK, et al. (2008) Blocking acid-sensing ion channel 1 alleviates Huntington’s

disease pathology via an ubiquitin-proteasome system-dependent mechanism. Hum

Mol Genet 17:3223–3235.34. He KL, et al. (2008) Endothelial cell annexin A2 regulates polyubiquitination and

degradation of its binding partner S100A10/p11. J Biol Chem 283:19192–19200.35. Jain RK, Munn LL, Fukumura D (2011) Transparent Window Models and Intravital

Microscopy: Imaging Gene Expression, Physiological Function and Therapeutic

Effects in Tumors. Tumor Models in Cancer Research, ed Teicher BA (Humana,

New York), pp 641–681.

Garkavtsev et al. PNAS | July 12, 2011 | vol. 108 | no. 28 | 11601

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0