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Dehydro-α-lapachone, a plant product with antivascular activity Igor 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; bHarvard School 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 for many malignancies. However, most of them target the VEGF pathway and lead to refractoriness. To improve the diversity of options for antivascular therapy, we applied a high-throughput screen for small molecules targeting cell adhesion. We then assayed the resulting antiadhesion hits in a transgenic zebrafish line 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 pruning and growth delay in orthotopic mammary tumors in mice. We show that DAL targets cell adhesion by promoting ubiquitination of the Rho-GTPase Rac1, which is frequently up-regulated in many different 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 bacterial infections (1, 2). Antiangiogenic agents, most of which target VEGF or its receptors, are emerging as standard therapies for several 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). Considering these limitations, the development of antivascular agents that target 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 in tumors are abnormal, featuring unusual leakiness, high tortuos- ity, and inefficient network structure (10). The immature ECs of this abnormal neovasculature in tumors have weak cell-cell junctions (10–13)—likely making them particularly sensitive to antiadhesion therapies. Candidate antiadhesion agents for anti- vascular therapy have been identified and have shown promising results in preclinical studies (14). Unfortunately, these early at- tempts at development of antiadhesion agents for antivascular therapy were met with failure stemming from issues with toxicity. We hypothesized that a nontoxic compound targeting cell adhesion would produce a safe antivascular effect specific to neovasculature. To this end, we devised a high-throughput cell adhesion screen to identify candidate nontoxic antiadhesion compounds with anti- vascular activity in tumors.
Results and Discussion Screening Yields a Natural Product—DAL. We developed a unique screening strategy to identify potential agents that target adhe- sion of ECs or cancer cells to their substrate. To this end, we began by screening 50,000 compounds in a high-throughput manner (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. Cell adhesion adaptor proteins have a domain for binding actin fila- ments (15, 16), thus adhesion molecules are directly linked to the actin cytoskeleton. We reasoned that the agents that affect cell adhesion may be monitored through remodeling of actin fila- ments. As our second screening step, we explored the effect of the selected compounds on actin assembly and redistribution by fixing and staining-treated cells with phalloidin. We observed changes in actin assembly and cell shape after treatment with 12 compounds. We next prioritized this set for compounds that were nontoxic to normal cells, cancer cells, and zebrafish. Of these 12 compounds, two were selected based on the in vitro and in vivo assessment. One of these compounds, dehydro- α-lapachone (DAL; Fig. 1B), showed structural similarities to β-lapachone, which has demonstrated antitumor and anti- trypanosomal activities through DNA topoisomerase I in- hibition and prevention of DNA repair (17, 18). We further tested DAL for toxicity in SCID mice, and found that mice are tolerant to i.p. doses of up to 100 mg/kg with no signs of tox- icity. We therefore selected DAL—a natural product from the Tabebuia Avellaneda tree—for further study.
DAL Shows Antivascular Effects in Zebrafish Models. To elucidate the potential impact of DAL on the process of vascular network formation, we next assessed its effects in zebrafish embryos at different stages of development. To visualize vessel defects in detail, we used transgenic fish expressing EGFP in endothelial cells (Tg(fli1:EGFP)y1) (19, 20). This model expresses EGFP in blood vessel ECs throughout normal development and during fin regeneration (21). We first characterized the effects of DAL on normal vascular development in zebrafish embryos. In control embryos, developing vessels migrated from the lateral plate mesoderm to the midline, where they coalesced into a vascular cord. These endothelial clusters subsequently established the pattern of the dorsal aorta and posterior cardinal vein. Inter- somitic vessels sprouted at designated branch sites in control embryos after dorsal aorta formation. In contrast, after treat- ment with DAL, the sprouting of intersomitic vessels at their designated 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. analyzed data; 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 from these companies was used in these studies. There is no significant financial or other competing interest in the work.
*This Direct Submission article had a prearranged editor. 1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
11596–11601 | PNAS | July 12, 2011 | vol. 108 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1104225108
treatment resulted in a profound reduction in the complexity of the arterial network. These findings demonstrate that DAL can interfere with the regulation of vascular formation and branching morphogenesis during development. Interestingly, DAL did not show any effects on the vasculature in adult fish, probably due to the stable junctions and postmitotic nature of the mature vasculature. To examine whether DAL has a similar antivascular effect on
neovasculature in adult zebrafish, we induced neovascularization by amputating the caudal fin at the ≈50% proximal–distal level and imaged fin rays and vessels from each fish over time. In control zebrafish caudal fins, amputated blood vessels healed their ends by 24 h after amputation and then reconnected arteries and veins through anastomosis, with blood flow resuming at wound sites by 48 h after amputation (Fig. 2B). Meanwhile, regenerating vessels in fish treated with DAL had defects in anastomosis and plexus formation. All control fish developed normal blood vessels and formed anastomotic bridges at the amputation plane by 9 d after amputation; in contrast, we did not 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 for neovasculature, we sought to determine the effects of DAL on tumor vasculature in mammals. To study these effects quanti- tatively, we conducted fluorescent angiographies in female SCID mice bearing orthotopic 4T1 mammary tumors in mammary fat pad windows via intravital multiphoton microscopy (Fig. 3A) (22–24). We treated these mice with 37.5 mg/kg DAL or saline daily for 5 d by i.p. injection. We found that DAL treatment decreases 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 tumor vascular length (normalized to tumor volume) versus saline treatment (Fig. 3C; P = 0.007, day 2; P = 0.02, day 4), whereas mean tumor vascular diameter remains the same (Fig. 3D).
These data indicate that DAL reduces vascular density in tumors through vessel pruning.
DAL Inhibits T