Orally administered lenalidomide (CC-5013) is anti-angiogenic in vivo and inhibits endothelial cell migration and Akt phosphorylation in vitro
Introduction
Angiogenesis, new blood vessel formation from existing vasculature, plays a crucial role in tumor growth and progression in a variety of human neoplasms (Folkman, 1971). New blood vessels are required for tumors to grow beyond 1–2 mm3 or metastasize and so inhibition of the angiogenic process has become an important strategy for the treatment of cancer (Carmeliet and Jain, 2000). Thalidomide has been shown to inhibit angiogenesis in different experimental models in vivo (although thalidomide is anti-angiogenic in rabbits only when co-incubated with rabbit or human microsomes due to the species-specific nature of its anti-angiogenic activity) and for this reason has been investigated as an anti-cancer agent (D'Amato et al., 1994, Kenyon et al., 1997). Indeed, the success of thalidomide in the treatment of patients with multiple myeloma (Singhal et al., 1999) has led to its use in the treatment of a variety of other cancers.
The Immunomodulatory Drug (IMiD®) lenalidomide is a structural analogue of thalidomide whose mechanism(s) of action remains unknown (Muller et al., 1996, Muller et al., 1999). Like thalidomide, lenalidomide has shown efficacy in patients with relapsed multiple myeloma (MM) (Bartlett et al., 2004a, Richardson et al., 2002). In this regard, we have previously shown that lenalidomide inhibits angiogenesis in vitro in both rat and human system, and is able to induce significant tumor growth delay in a murine colorectal model of cancer (Dredge et al., 2002).
In the current study, we set out to test the ability of lenalidomide to inhibit angiogenesis in vivo since this has not yet been demonstrated. For this we utilized the rat mesenteric window assay (RNA). We also administered lenalidomide orally since this is relevant to its clinical application and also circumvents possible physical trauma caused by repeated ip injection which could influence angiogenesis in mesenteric windows. Induction of angiogenesis in the mesenteric windows was generated using bFGF as this growth factor may play a role in angiogenesis-dependent conditions including multiple myeloma (Bisping et al., 2003) and other cancers (O'Brien et al., 1997, Shiojima et al., 2002).
It was also apparent from our earlier studies that lenalidomide, while having little effect on growth factor induced human umbilical vein endothelial cell (HUVEC) proliferation, inhibits spontaneous EC migration (Dredge et al., 2002). The ability of endothelial cells to migrate and form capillary-like structures is critical in growth factor-induced angiogenesis. Furthermore, this process is dependent on signaling via PI3K-Akt-dependent pathways (Chavakis et al., 2001, Morales-Ruiz et al., 2000). We therefore set out to determine anti-migratory effects on HUVEC in response to relevant growth factors with a view to investigating a potential inhibitory effect on Akt activation in the mechanism of action of this novel compound.
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Animals
For the windows assay, adult male Sprague–Dawley rats (Harlan, UK) were acclimatized for 2 weeks to standardized environment. All procedures were carried out in accordance with UK Home Office (Scientific Procedures) Act 1986 and institutional guidelines. The ethical guidelines that were followed meet the standards set by the UKCCCR guidelines (Workman et al., 1998). Bodyweights were measured throughout the course of the study at weekly intervals.
For the PK study, jugular vein-cannulated male
Results
In the RMWA, representative differences between vehicle (Fig. 1a) and 50 or 250 mg/kg lenalidomide-treated rats (Figs. 1b and c, respectively) were visualized by staining with an antibody against rat endothelium in bFGF-induced angiogenic windows. PBS alone did not induce vessel formation and VEGF-induced vessel formation was of reduced magnitude compared to bFGF (data not shown). The induction of angiogenesis by bFGF was significantly inhibited by oral treatment of lenalidomide in a
Discussion
In this study, we have demonstrated for the first time that lenalidomide inhibits growth factor-induced angiogenesis in vivo and that this can be achieved by oral dosing. This follows on from previous work in which it was demonstrated that lenalidomide potently inhibits angiogenesis using in vitro models of angiogenesis and significantly reduces tumor growth rates in a murine colorectal cancer model (Dredge et al., 2002).
In the present study, we show that lenalidomide inhibits growth-factor
Acknowledgments
We thank Professor Klas Norrby (Gothenborg University, Sweden) and Faribourz Payvandi (Celgene Corporation) for helpful discussions. S.P.R. is the recipient of a Royal Society University Research Fellowship.
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Current address: Centre for Immunology and Cancer Research, Princess Alexandra Hospital, Woolloongabba, Brisbane 4102, Australia.