Mitochondria as targets in angiogenesis inhibition

Molecular Aspects of Medicine 31 (2010) 113–131

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Molecular Aspects of Medicine

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Review

Mitochondria as targets in angiogenesis inhibition

Danielle Park, Pierre J. Dilda *

Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney 2052, Australia

a r t i c l e i n f o

Article history:Received 23 June 2009Accepted 2 December 2009

Keywords:MitochondriaAngiogenesisAngiogenesis inhibitors

0098-2997/$ - see front matter � 2009 Elsevier Ltddoi:10.1016/j.mam.2009.12.005

* Corresponding author. Tel.: +61 2 93858796; faE-mail address: [email protected] (P.J. Dilda)

a b s t r a c t

Angiogenesis is integral to the growth and metastatic spread of tumours, and its targetingis an effective anti-tumour strategy. Currently hundreds of anti-angiogenic therapeuticsexist in varying stages of development, a number of which have recently gained US Foodand Drug Administration (FDA) approval for the treatment of various human cancers.One class of anti-angiogenic agents directly inhibit endothelial cell function and induceendothelial cell death so as to prevent their integration into new blood vessels. The mito-chondria are the focal point for a variety of pro-apoptotic signals, and this review high-lights those anti-angiogenic agents that involve the mitochondria in the execution ofendothelial cell death. A brief overview of angiogenesis and the mitochondrial apoptoticpathway is also given.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142. Tumour angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

2.1. Vascular endothelia growth factor (VEGF). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152.2. Fibroblast growth factors (FGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152.3. Matrix metalloproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152.4. Angiopoietins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162.5. Integrins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162.6. Thrombospondin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

3. Current anti-angiogenic therapies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164. The role of mitochondria in apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

4.1. Cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194.2. Smac/DIABLO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194.3. Apoptosis inducing factor (AIF) and Endonuclease G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194.4. Bax/Bak associated pores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194.5. Mitochondrial permeability transition pore (MPTP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

5. Angiogenic inhibitors targeting the mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.1. GSAO and derivatives as mitochondrial poisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205.2. Vitamin E analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1245.3. Inhibitors of survivin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1255.4. Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1255.5. Collagen derived angiogenic inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265.6. Silibinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1275.7. Neovastat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
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114 D. Park, P.J. Dilda / Molecular Aspects of Medicine 31 (2010) 113–131

6. Potential mitochondrial targets for the inhibition of angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
6.1. Prohibitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
1. Introduction

Angiogenesis – the process of new blood vessel formation; is prevalent during embryonic development but is highly re-stricted in the healthy adult. In a normal adult vessel only 0.01% of endothelial cells are dividing at any given time and assuch the quiescent adult vasculature has a very low rate of turnover (Engerman et al., 1967; Hobson and Denekamp,1984). Endothelial cells however retain the ability to divide rapidly, and are induced to do so in exceptional physiologicalscenarios such as would healing, menstruation and pregnancy. Angiogenesis is also induced in a number of pathologiesincluding rheumatoid arthritis and diabetic retinopathy (Carmeliet, 2005; Carmeliet and Jain, 2000; Folkman, 2007).

Angiogenesis has also been implicated in the growth and metastatic spread of tumours, and in 1971 it was hypothesizedthat inhibition of angiogenesis may be an effective anti-tumour strategy (Folkman, 1971). Since this time, extensive researchinto the underlying mechanisms of angiogenesis has revealed key angiogenic mediators such as growth factors, cytokinesand their receptors, that can be targeted by anti-tumour therapeutics. Currently there exist over 300 therapeutic angiogenicinhibitors in varying stages of development, of which 8 have recently gained US Food and Drug Administration (FDA) ap-proval for the treatment of various human cancers (Folkman, 2007).

It is now well established that a series of genetic mutations precede tumour formation, and that these modifications con-fer a selective growth advantage. Because numerous mutations are required for tumour progression it is argued that tumoursare inherently predisposed to genetic mutation, and are thus ‘genetically unstable’. This may be an outcome of defective DNArepair within the tumour cell, which in turn facilitates oncogene activation or silencing of tumour suppressor genes vianucleotide substitution/insertion or deletion, or chromosomal modifications (Lengauer et al., 1998; Sieber et al., 2003).The enhanced rate of genetic mutation in tumour cells may also confer resistance toward anti-cancer therapeutics, the tu-mour evading treatment by adapting and bypassing the targeted pathway.

One of the attractions of anti-angiogenic therapies is their potential to circumvent tumour acquired drug resistance bytargeting genetically stable endothelial cells. In addition, direct angiogenic inhibitors that target proliferating and/or migrat-ing endothelial cells, may be more effective than indirect inhibitors that target the release of angiogenic factors from tu-mours. In the case of the latter, the genetically unstable tumour may simply upregulate other pro-angiogenic factors inresponse to treatment, and so evade therapy.

Direct inhibitors of endothelial cell function generally induce cell death, preventing them from proliferating and integrat-ing into a new vessel. The mitochondria are the focal point for a variety of pro-apoptotic signals and of particular interest tothis review are those anti-angiogenic agents that employ the mitochondria in the execution of endothelial cell death. Thisgroup of compounds involve agents that directly target the mitochondria such as GSAO and Vitamin E analogues, and thosethat initially target other cellular processes but involve the mitochondria downstream. An overview of tumour angiogenesisand the mitochondrial apoptotic pathway is given to assist the reader in understanding the mechanisms of action of theseanti-angiogenic compounds.

2. Tumour angiogenesis

The ability of tumours to acquire their own blood supply is one of the key molecular events dictating malignant growth.Most adults possess dormant microscopic tumours but only �1/600 of these switch to an angiogenic phenotype and becomedetectable in the host (Folkman and Kalluri, 2004). The dormant tumour can reach around 1 mm3 before its growth is limitedby the amount of nutrients it can obtain from the pre-existing vasculature. The tumour must therefore acquire the ability totrigger sprouting of nearby blood vessels, which infiltrate the tumour and allow further growth. The newly formed vascu-lature also allows the tumour to metastasize (Folkman, 2006a).

Tumour angiogenesis is initiated when the balance between pro and anti-angiogenic factors shifts, favouring the former.Various environmental changes such as oxygen deprivation, hypoglycaemia or low pH initiate this, as do common tumouri-genic mutations such as oncogene activation or silencing of tumour suppressor genes (Kerbel, 2008). H-ras, v-Src and HER2oncogenes for instance have all been associated with upregulation of pro-angiogenic mediators (Arbiser et al., 1997; Mukho-padhyay et al., 1995; Petit et al., 1997; Rak et al., 1995). Tumour hypoxia similarly activates the hypoxia inducible factor-1(HIF-1) class of transcription factors shifting the tumour toward a glycolytic metabolism and upregulating a host of pro-angiogenic factors. Under normoxic conditions HIF-1 is targeted for degradation by the von Hippel-Lindau protein, but dur-ing hypoxia HIF-1 is unaffected and active (Safran and Kaelin, 2003).

Extensive research into the underlying mechanisms of angiogenesis has revealed a number of angiogenic factors thatregulate key aspects of tumour neovascularisation. They each govern varying stages in the angiogenic process. One of theearliest events is degradation of the extracellular matrix (ECM) by proteases. These matrix degrading enzymes are produced

D. Park, P.J. Dilda / Molecular Aspects of Medicine 31 (2010) 113–131 115

by tumour cells, stromal cells and endothelial cells and via degradation of the vascular basement membrane liberate asso-ciated endothelial cells and sequestered growth factors. Growth factors such as vascular endothelial growth factor (VEGF) inturn stimulate endothelial cell proliferation and migration, and endothelial cells navigate their way to a provisional matrixforming the lumen of an intermediate vessel. In the later stages of angiogenesis a new basement membrane is assembled, theimmature vessel remodelled by angiopoietins and stabilized by recruitment of pericytes (Kalluri, 2003).

These angiogenic mediators are considered the gate keepers of varying steps in the angiogenic process, and as such rep-resent attractive anti-angiogenic targets. A brief overview of the central players and their mechanism of action is given.

2.1. Vascular endothelia growth factor (VEGF)

The VEGF family of growth factors is composed of series of structurally related molecules including VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF). VEGF-A (often simply referred to as VEGF) is the best charac-terized of these, and has four splice variants of 121, 165, 189 and 206 amino acids. Whilst VEGF121 is freely secreted, VEGF189

and VEGF206 are ECM bound. The dominant isoform VEGF165 is both partially secreted and sequestered in the ECM (Ferrara,2004).

VEGF-A is secreted predominantly by the tumour and the associated stroma, but is also released from the ECM upon pro-teolytic degradation by matrix metalloproteinases (MMPs). VEGF-A mostly functions in paracrine fashion, signalling throughreceptor tyrosine kinases on the surface of endothelial cells. Two such receptors have been identified, VEGF receptor 2 (VEG-FR-2, also known as Flk-1 or KDR) which mediates most of the downstream effects of VEGF-A, and VEGFR-1 (Flt-1). VEGF-Ahas also been shown to bind neuropilins 1 and 2, which likely act as co-receptors for VEGFR-1 and VEGFR-2 by enhancing thebinding affinity of the receptor for its ligands. VEGFR-2 is upregulated on the surface of angiogenic endothelial cells, andbinding by VEGF-A stimulates a cascade of signalling pathways culminating in increased vascular permeability, endothelialcell survival, proliferation and migration (Hicklin and Ellis, 2005; Kerbel, 2008).

Increased vascular permeability is one of the earliest processes in angiogenesis, resulting in the leakage of platelets andfibrinogen to the extra-vascular space (Dvorak, 2002; Hicklin and Ellis, 2005). The interaction of platelets with the underly-ing lamina results in discharge of their a granules, and release of pro-angiogenic proteases, lipases, and growth factors suchas VEGF and platelet derived growth factor (PDGF) (Cheresh and Stupack, 2008). Fibrin deposits in the tumour stroma alsopromote a pro-angiogenic environment (Dvorak, 2002). VEGF-A is thought to increase vascular permeability via the activa-tion of Src kinases. This signalling pathway induces endothelial cell contraction, as well as disrupting cell–cell junctions byphosphorylating VE-cadherin (Cheresh and Stupack, 2008). VEGF-A may also promote vascular permeability by stimulatingphospholipase C/diacylglycerol leading to an influx of calcium and production of nitric oxide (Bates and Harper, 2002).

Another prominent function of VEGF-A is to induce endothelial cell proliferation. This is primarily executed by membersof the mitogen activated protein kinase (MAPK) family. For instance, VEGF-A has been shown to activate extracellular reg-ulated kinases 1 and 2 (Erk1/Erk2) via the Ras signalling pathway, leading to the phosphorylation of transcription factors andproteins controlling cell cycle progression (Meadows et al., 2001). VEGF is also known to activate the c-Jun N-terminal kinase(JNK) (Hicklin and Ellis, 2005).

The mechanisms by which VEGF-A induces endothelial cell migration are not as transparent. VEGF-A dependant activa-tion of p38 MAP kinase and focal adhesion kinase (FAK) have been implicated in reorganization of the actin cytoskelton,whilst VEGF production of nitric oxide may navigate the non-chemotactic scalar movement (podokinesis) of endothelial cells(Hicklin and Ellis, 2005).

Finally VEGF-A also promotes endothelial cell survival via inhibition of apoptosis. VEGF-A has been shown to upregulatethe anti-apoptotic Bcl-2 and A1 proteins (Gerber et al., 1998) as well as the apoptotic inhibitors XIAP and survivin (Tran et al.,1999). VEGF-A also activates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway which is involved in a whole spectrum ofanti-apoptotic signalling discussed in detail later in this review.

2.2. Fibroblast growth factors (FGF)

There are 22 structurally related members of the FGF family. A number of these have been implicated as angiogenic medi-ators, however the bulk of experimental data pertains to the pro-angiogenic role of FGF1 and FGF2 (also known as bFGF).FGFs are secreted by tumour cells, the associated stroma and released from the ECM upon proteolytic degradation by MMPs.They are involved in almost every step of the angiogenic program, and exert their affects by binding to tyrosine kinase recep-tors on the surface of endothelial cells. In the early stages of angiogenesis, FGFs promote endothelial cell detachment andmigration by regulating expression of cadherins and integrins and so disrupting cell–cell junctions. FGFs have also beenshown to upregulate various MMPs in endothelial cells facilitating degradation of the basement membrane. Endothelial cellproliferation is then induced by FGFs via activation of the MAPK and protein kinase C (PKC) signalling pathways, and in thelater stages of angiogenesis FGFs stabilize the cellular adhesions required for vessel maturation (Presta et al., 2005).

2.3. Matrix metalloproteinases

Matrix metalloproteinases are a family of 23 zinc dependant enzymes that facilitate endothelial cell migration duringangiogenesis via degradation of the ECM. They can be divided into six categories according to their structure and substrate


specificity, collagenases, gelatinases, stromelysins, matrilysins membrane-type MMPs, and other unclassified MMPs. Expres-sion of most MMPs is low in normal tissues, but is strongly upregulated during tumour angiogenesis in response to angio-genic growth factors (such as VEGF and bFGF) or oncogene activation. MMPs are secreted by endothelial cells, tumours cellsand infiltrating inflammatory cells (Rundhaug, 2005).

MMP degradation of the vascular basement membrane and other ECM components is essential to endothelial cell migra-tion; however MMPs are also involved in numerous other pro-angiogenic events. MMPs facilitate release of growth factorssequestered in the ECM, as well as cleaving endothelial cell–cell adhesions – thereby increasing vascular permeability. MMPshave also been implicated in pericyte detachment from the parent vessel (Raffetto and Khalil, 2008).

Excessive proteolysis is undesirable in angiogenesis as it results in destruction of the provisional matrix scaffold support-ing endothelial cell invasion. MMPs are therefore regulated at a number of levels. MMPs are synthesized as latent zymogenpre-cursors and must undergo proteolysis in order to be activated. They are also regulated at the level of transcription, andby Tissue Inhibitors of Metalloproteinases (TIMPs) which bind directly to MMPs and so inactivate them (Raffetto and Khalil,2008).

Interestingly MMPs have also been implicated as negative regulators of angiogenesis, as digestion of the vascular base-ment membrane leads to release of fragmented ECM proteins which act as endogenous angiogenic inhibitors. The proteolysisof Types XVIII and IV collagen for instance results in the formation of at least four inhibitors: endostatin (the C-terminal non-collagenous 1 (NC1) domain of Type XVIII collagen a1 chain), tumstatin (Type IV collagen a3 chain NC1), arrestin (Type IVcollagen a1 chain NC1) and canstatin (Type IV collagen a2 chain NC1), whilst MMP digestion of plasminogen results in theangiogenic inhibitor angiostatin. The inhibitory actions of these ECM fragments are largely mediated by integrin binding, andbroadly speaking result in inhibition of endothelial cell proliferation and endothelial cell apoptosis. The latter is discussed inmore detail further in the review.

2.4. Angiopoietins

Together with the VEGF receptors, only one other class of receptor tyrosine kinases, Tie-1 and Tie-2, are largely endothe-lial specific. These receptors and their ligands, angiopoietins 1 and 2 (Ang-1 and Ang-2), play a significant role in both thevery earliest and latest stages of angiogenesis. Ang-1 binds the Tie-2 receptor, and is antagonized by Ang-2. One of the pri-mary functions of Ang-1 in the normal adult vasculature is to maintain vessel quiescence via pro-survival signalling, smoothmuscle cell recruitment and inhibition of endothelial cell permeability. During the early stages of tumour development,however, Ang-2 is upregulated in endothelial cells and destabilizes the parent vessel by antagonizing Ang-1. This sets in mo-tion the angiogenic cascade. In the later stages of angiogenesis, Ang/Tie signalling acts in concert with VEGF to stabilize andmature vessels (Augustin et al., 2009).

2.5. Integrins

The integrins are a family of cell surface receptors that mediate adhesion to the ECM. They are composed of a and b sub-units (of which there are 18 and 8, respectively) which associate to form 24 unique heterodimers. A number of endothelialcell integrins have been implicated in angiogenesis, including a1b1, a2b1, a4b1, a5b1, a6b1, a6b4, a9b1, avb3 and avb5, many ofwhich are upregulated in response to angiogenic growth factors such as bFGF and VEGF. Integrins do not have any intrinsicenzymatic activity, but upon ligation with the ECM cluster in a focal adhesion complex with protein kinases such as Src andFAK, as well as signalling intermediates. These in turn activate MAPK and phosphatidylinositol 3-kinase (P13K) signallingpathways culminating in endothelial cell migration, proliferation and survival (Avraamides et al., 2008).

2.6. Thrombospondin

Thrombospondins are a family of five extracellular glycoproteins found in the extracellular matrix. Thrombospondin-1(Tsp-1) was the first protein to be identified as a naturally occurring angiogenic inhibitor, although Tsp-2 has also beenimplicated. Both Tsp-1 and Tsp-2 are known to inhibit angiogenesis by hindering endothelial cell proliferation and migra-tion, and inducing endothelial cell apoptosis. The former is achieved via downregulation of VEGFR-2 on endothelial cells thusantagonizing the pro-migratory signalling of VEGF-A, whilst the latter is induced by upregulation of the Fas ligand. Bothfunctions appear to be mediated through interaction of Tsp-1 and Tsp-2 with CD36 (Zhang and Lawler, 2007).

3. Current anti-angiogenic therapies

Pharmaceutical intervention of the functions of pro-angiogenic factors has successfully brought the first anti-VEGF drug,Avastin, to the market for the treatment of human cancers after demonstrating a survival benefit in a clinical Phase III met-astatic colorectal study (Hurwitz et al., 2004). This success has validated the early concept that tumour growth is dependenton angiogenesis, and that anti-angiogenesis is a valid approach for not only cancer therapy (Folkman, 1971) but also for otherangiogenesis-dependent diseases such as autoimmune diseases, age-related macular degeneration and atherosclerosis.Although it remains to be clarified to what extent treating abnormal angiogenesis in different angiogenesis-dependent


diseases will be successful, the recent approval of Ranibizumab (Lucentis), a fragment of Bevacizumab (Avastin), for age-re-lated macular degeneration suggests that such strategies should be valid and merit investigation. The process of angiogen-esis can then be considered an ‘organizing principle’ underlying different diseases (Folkman, 2007). In consequence, thedevelopment of therapeutics for one disease could aid the development of therapeutics for others.

Today, over 20 angiogenic growth factors and over 300 anti-angiogenic molecules targeting different signalling pathwaysare being tested for their anti-cancer efficacies at preclinical and clinical stages. Eight new drugs in which anti-angiogenicactivity is considered to be central to their therapeutic effects have been approved by the FDA in the United States forthe treatment of cancer (Table 1). Many other drugs that have varying degrees of anti-angiogenic activity are currently inclinical trials. These drugs have various targets such as HIF-1a, b tubulin, avb3, avb5, a5b1 and a2 integrins, VE-cadherin,Tsp-1, matrix metalloproteinase or histone deacetylase (for review see Folkman, 2007). In addition, several previously ap-proved drugs for the treatment of cancer and non-malignant diseases revealed anti-angiogenic activity on top of anti-canceractivity. Examples include Celecoxcib (a cyclooxygenase 2 (COX-2) inhibitor), Erlotinib (Tarceva, a growth factor receptorinhibitor), Bortezomib (Velcade, a proteosome inhibitor) (Yasui et al., 2006), and Interferon-a (Folkman, 2006b).

The principal regulators of angiogenesis are the VEGFs and their receptors. Owing to its central role in promoting tumourgrowth, VEGF-A has become a key therapeutic target and its functions can be blocked at different levels of the signallingpathways (Cao, 2008). Currently, the majority of FDA-approved angiogenesis inhibitors, as well as those in Phase III clinicaltrials, neutralize VEGF, target its receptor or suppress its expression by tumour cells.

The first anti-VEGF-A cancer drug, Avastin (Bevacizumab) is a humanized anti-VEGF neutralizing antibody that blocks theinteraction of VEGF-A with its receptors (Hurwitz et al., 2004). Avastin has a relatively long half-life in the blood stream andwas approved by the US FDA in February 2004 for use in combination with standard chemotherapy for late-stages colon,breast and lung cancers. In these applications the drug appears to prolong life for up to a few months. Avastin is alreadyone of the world’s biggest selling cancer drugs, with sales of $2.7 billion in the United States alone last year. Encouragingreports are also emerging from the use of Avastin and Paclitaxel in previously untreated patients with metastatic breast can-cer (Miller, 2003). The use of Avastin in early-stage cancers however has not had the same success. Genentech recently an-nounced that the treatment of early-stage (adjuvant) colon cancer with Avastin and chemotherapy did not reach the primaryendpoint in a Phase III clinical trial (American Society of Clinical Oncology meeting, May 2009). This was the first trial of Ava-stin in early-stage cancer and results do not affect approved indications in advanced (metastatic) disease. Aptamers (Pegap-tinib and Ranibizumab), which could be considered fragments of Avastin, can also interfere in the interaction of VEGF-A andits receptors. In randomized clinical trials, Ranibizumab injected into the eye at monthly intervals showed dramatic successin patients with age-related macular degeneration. In 2004, Pegaptanib (Macugen) was the first anti-VEGF drug to be ap-proved by the FDA for the treatment of age-related macular degeneration (Gragoudas et al., 2004). Soluble VEGFRs (lackingtransmembrane and intracellular signalling domains) that trap VEGF-A are also currently under evaluation (Holash et al.,2002; Wu et al., 2006).

Another approach of anti-VEGF therapies is to target the VEGF receptors and their signalling pathways. This has beenachieved by high affinity antibodies directed against human VEGFR-1 or VEGFR-2 (Jain et al., 2006; Wu et al., 2006) and by anumber of small chemical compounds. These anti-angiogenic agents bind to VEGFRs and so inhibit their tyrosine kinase activity.

As discussed previously, the expression of VEGFRs is largely confined to endothelial cells, although a number of otherwidely expressed receptor tyrosine kinases (RTK) also mediate angiogenesis. Inhibitors of RTKs that are more broad spec-trum in their approach are therefore likely to be of greater benefit than specific inhibitors. For example, Semaxanib and Vat-alanib which specifically inhibit VEGFRs have shown promising results in animal studies, but have not produced a benefit inclinical trial and consequently been terminated. Anti-angiogenic agents that inhibit a range of tyrosine kinase receptors weremore successful. Sunitinib (Sutent) (Motzer et al., 2007) which acts by inhibiting signalling pathways downstream of a num-ber of receptors (VEGFR-2, PDGFR-b, KIT, and FLT-3 kinases), has been approved for the treatment of advanced renal cell car-cinoma. A Phase III trial study showed that Sunitinib produced unexpectedly positive results for prolonging survival inpatients with renal carcinoma. This drug is also used in imatinib–mesylate-resistant gastrointestinal stromal tumours (Mot-zer et al., 2007). Sorafenib (Nexavar), also a multi-kinase inhibitor (Escudier et al., 2007) has been licensed for late-stage re-nal cell carcinoma as second-line therapy. In patients with advanced hepatocellular carcinoma, median survival and the timeto radiologic progression were nearly 3 months longer for patients treated with Sorafenib than for those given placebo (Llo-vet et al., 2008).

Table 1Anti-angiogenic therapeutics FDA approved for the treatment of cancer.

Angiogenic inhibitor Target FDA approved for

Avastin/bevacizumab (Genentech) VEGF Non-small cell lung carcinoma, metastatic colorectal cancer and breastcancer

Cetuximab/Erbitux (Bristol-Myers SquibbImClone)

EGFR Metastatic colorectal carcinoma and head and neck cancer

Panitumumab/Vectibix (Amgen) EGFR Metastatic colorectal carcinomaErlotinib/Tarceva (Genentech OSI Roche) EGFR Non-small cell lung carcinoma and pancreatic cancerSunitinib/Sutent (Pfizer) Multi-kinase inhibitor Advanced renal cell carcinoma and gastrointestinal tumoursSorafenib/Nexavar (Bayer Onyx) Multi-kinase inhibitor Advanced renal cell carcinoma and advanced hepatocellular carcinoma


The cancer drugs Erlotinib (Tarceva), Gefitinib (Iressa), Vandetanib (Zactima) and the monoclonal antibody Cetuximab(Erbitux) were originally developed as inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase. Gefitinib,Vandetanib and Cetuximab were subsequently shown to inhibit the VEGF receptor. Erlotinib is a highly selective tyrosinekinase inhibitor that is approved for the treatment of patients with locally advanced or metastatic non-small cell lung car-cinoma (NSCLC) after failure of at least one prior chemotherapy regimen (Herbst and Sandler, 2008). It is also approved incombination with Gemcitabine for the first-line treatment of patients with locally advanced, unresectable, or metastatic pan-creatic cancer (Duffy et al., 2008). Another tyrosine kinase inhibitor, Gefitinib was also approved as a third-line option forpatients with NSCLC. A large Phase III trial in patients with pretreated advanced NSCLC demonstrated the non-inferiorityof Gefitinib in comparison with Docetaxel for overall survival together with improved tolerability profiles. As a result, Gef-itinib is expected to have a large impact in the management of patients with NSCLC (Reck, 2009). The monoclonal antibodyCetuximab (Erbitux) produces an anti-tumour effect in vivo that is due to the direct inhibition of the EGFR-dependent mito-genic pathway and in part to the inhibition of secretion of VEGF (Morelli et al., 2006).

Suppressing VEGF expression by tumour cells is another means of blocking the angiogenic process. Vascular endothelialgrowth factor gene silencing via VEGF-siRNA can effectively inhibit the production of VEGF and exert an anti-angiogenic andanti-tumourigenic effect both in vitro and in vivo (He et al., 2009; Wang et al., 2008). Gene therapies designed to block VEGF-A secretion in tumour cells also present an attractive approach for suppression of tumour growth and tumour-induced vas-cular hyperpermeability (Li et al., 2006; Lin et al., 2008). In a Phase I clinical study VEGF antisense was well tolerated, withbiologic effects and preliminary evidence of clinical efficacy (Levine et al., 2006).

Although the results of these clinical trials are encouraging as proof of principle, the effects are undeniably modest. Clin-ical practice reveals that therapy with angiogenesis inhibitors often does not prolong survival of cancer patients for morethan months, because tumours elicit evasive resistance. One difficulty with anti-angiogenic therapies is that the hypoxic tu-mour microenvironment may select for tumour cells populations that are less dependent on angiogenesis and resistant toanti-angiogenic therapy. Typical tumourigenic mutations like that of the Tp53 gene are associated with resistance to hypox-ia, and the hypoxic environment created by anti-angiogenic agents likely selects for continued growth (Yu et al., 2002).

Hypoxic selection of tumour cells may explain two recent studies in which treatment of tumours with Sunitinib and aVEGFR-2 function-blocking antibody (DC101) in mice was accompanied by tumour resistance (Ebos et al., 2009; Paez-Ribeset al., 2009). Although these anti-VEGF therapies reduced primary tumour growth, both were shown to enhance tumourinvasiveness and consequently tumour metastasis. It is possible that disruption of the tumour vasculature by means of cre-ating a hypoxic environment selected for a resistant tumour phenotype with greater malignant potency (Loges et al., 2009;Paez-Ribes et al., 2009).

Tumours may also evade anti-angiogenic therapies by upregulating other pro-angiogenic mediators and thus circumvent-ing the original target. This is particularly relevant to anti-angiogenic therapies that block only a single pathway, such asinhibitors of VEGF-A.

The genetic instability of tumour cells leads to a shift in the expression of pro-angiogenic factors so that the targetedangiogenic mediator becomes redundant. For example, treatment with anti-VEGFR-2 antibodies has been shown to upreg-ulate bFGF (Casanovas et al., 2005) whilst treatment with Bevacizumab has been associated with an increase in circulatingPlGF (Nissen et al., 2007). VEGF is also upregulated in response to anti-EGFR therapies (Bianco et al., 2005; Viloria-Petit andKerbel, 2004). Activation of alternate pro-angiogenic pathways therefore serves as an escape mechanism for the tumour. Be-cause of this, anti-angiogenic therapies that target only a single pathway would be most effective when used in combinationwith other anti-angiogenic agents. Anti-angiogenic therapeutics that are broad spectrum in their approach are also likely tobe more successful.

This area of tumour resistance may also be avoided by anti-angiogenic agents that target the tumours blood vessels di-rectly. Instead of targeting pro-angiogenic mediators that can be circumvented by the tumour, this class of compounds di-rectly inhibit the endothelial cells of new blood vessels. This not only offers a reduction in tumourigenic blood vessels butalso eliminates the growth factors propagated by endothelial cells that assist in tumour proliferation. In many cases theseanti-angiogenic agents interfere with endothelial cell function and/or induce endothelial cell apoptosis. Of particular interestto this review are those anti-angiogenic agents that employ the mitochondria in the execution of endothelial cell death. Therole of mitochondria in apoptosis will now be discussed.

4. The role of mitochondria in apoptosis

Apoptosis is a programmed form of cell death essential to the elimination of superfluous or damaged cells in a range ofphysiological scenarios. Unlike necrosis, apoptosis involves the controlled packaging of cellular constituents into membraneenclosed vesicles (apoptotic bodies) and removal by phagocytes without eliciting a full immune response. One of the earliestevents characterizing apoptosis is a reduction in cell size due to an efflux of water, followed by membrane blebbing. As theapoptotic process proceeds, phosphatidyl serine is externalized in the outer plasma membrane leaflet, the mitochondrialmembrane potential is lost, nuclear chromatin is condensed and the DNA fragmented (Taylor et al., 2008).

Various apoptotic pathways have been characterized; and common to each of these pathways is the end-point activationof a group of cysteine proteases called caspases. Caspases, so named for their cleavage of substrates after an aspartate res-idue, can be classified as either initiator or executioner caspases. Whilst the role of the former (caspases 8, 9 and 10) is to


activate other caspases by cleavage, the latter (caspases 3, 6 and 7) are responsible for the degradation of cellular constitu-ents (Kroemer et al., 2007). This involves the cleavage of DNA into discrete fragments, activation of other pro-apoptotic pro-teins, and modification of the cytoskeleton resulting in membrane blebbing, and re-organisation of the plasma membranearound organelles to form apoptotic bodies. The executioner caspases also activate proteins responsible for phosphatidyl ser-ine exposure in the plasma outer membrane, which in turn permits phagocytic clearage of the apoptotic bodies (Taylor et al.,2008).

Depending on the stimulator of apoptosis, the caspases can be activated through the intrinsic (mitochondrial mediated)or extrinsic (death receptor mediated) apoptotic pathways. In the extrinsic pathway, death receptors on the plasma mem-brane such as tumour necrosis factor receptor 1 (TNFR1), Fas/CD95, TNF-related apoptosis inducing ligands 1 and 2 (TRAILs 1and 2) and translocating chain-association membrane protein (TRAMP) are bound by ligands leading to the formation of thedeath inducing signalling complex (DISC) and activation of caspase 8. This in turns leads to the activation of executionercaspases (Danial and Korsmeyer, 2004).

The anti-angiogenic agents described in this review induce endothelial cell death by targeting the mitochondria and soinducing the mitochondrial mediated pathway of apoptosis. This pathway is characterized first and foremost by permeabil-isation of the mitochondrial membrane and release of pro-apoptotic factors cytochrome c, apoptosis inducing factor (AIF),Endonuclease G, and second mitochondrial-derived activator of caspase/direct inhibitor of apoptosis binding protein withlow pI (Smac/DIABLO). Each of these pro-apoptotic factors have different cellular targets, however their cumulative releaseis considered ‘the point of no return’ in the apoptotic program (Kroemer et al., 2007).

4.1. Cytochrome c

Cytochrome c is perhaps the most well defined mitochondrial mediator of apoptosis. It resides within the intermembranespace largely tethered to cardiolipin in the inner-mitochondrial membrane and is released upon disruption of the cardiolipininteraction and permeabilisation of the mitochondrial membrane. Reactive oxygen species (ROS) have been shown to oxidizecardiolipin and so disrupt the electrostatic interactions confining cytochrome c to the membrane (Ott et al., 2002). Free cyto-chrome c in turn also mediates cardiolipin oxidation, working in a positive feedback loop to amplify its own release (Kaganet al., 2005). Once in the cytosol, cytochrome c forms a multi-protein complex with apoptosis protease activating factor-1(APAF-1) and ATP/dATP called the ‘apoptosome’ and initates the activation of caspase 9 and consequently downstream exe-cutioner caspases. It also amplifies mitochondrial membrane permeabilisation by discouraging calcium retention within theendoplasmic reticulum, leading to calcium mediated opening of the mitochondrial permeability transition pore (discussed ingreater detail below) (Boehning et al., 2003, 2004).

4.2. Smac/DIABLO

Smac/DIABLO is a 23 kDa protein that is confined to the intermembrane space of the mitochondria and upon release tothe cytosol binds to endogenous inhibitors of apoptosis (IAPs). Smac/DIABLO has been shown to neutralize IAP’s such asXIAP, cIAP1 and cIAP2, survivin and Apollon and so promote caspase activation (Vaux and Silke, 2003).

4.3. Apoptosis inducing factor (AIF) and Endonuclease G

AIF is a 62 kDa protein that is found tethered to the inner membrane of the mitochondria, or in a soluble form in the inter-membrane space. Upon permeabilisation of the mitochondrial membrane AIF translocates to the nucleus where it plays amajor role in chromatin condensation and DNA fragmentation. In normal physiological conditions AIF is known to detoxifyreactive oxygen species and maintain the respiratory chain Complex I (Vahsen et al., 2004). The release of AIF from the inter-membrane space therefore promotes apoptosis by introducing a defect in oxidative phosphorylation as well as removing cel-lular protection from oxidative stress (Modjtahedi et al., 2006).

Like AIF, Endonuclease G is a mitochondrial bound enzyme that translocates to the nucleus upon permeabilisation of themitochondrial membrane. It is similarly responsible for DNA fragmentation (Kroemer et al., 2007).

Each of these mitochondrial proteins play a role navigating the final stages of intrinsic apoptosis, however their functionis wholly dependent on mitochondrial membrane permeabilisation. In the healthy cell the outer mitochondrial membrane ispermeable only to solutes of up to 5 kDa via the voltage dependant anion channel (VDAC), whilst the inner membrane isnearly impermeable to all ions, allowing formation of a proton gradient that drives ATP synthesis. The function of most stim-uli of the intrinsic apoptotic pathway is thus to change membrane permeability (Kroemer et al., 2007). This is predominantlyachieved via the introduction of Bax/Bak associated pores or initiation of the mitochondrial permeability transition, both ofwhich will be now discussed in detail.

4.4. Bax/Bak associated pores

Bax and Bak are members of the multi-domain pro-apoptotic Bcl-2 family of proteins that each contains 3 Bcl-2 homologydomains. Whilst Bax is predominantly found in the cytosol or tethered to intracellular membranes, Bak is already embeddedin the outer mitochondrial membrane. Both proteins exert their pro-apoptotic affect by undergoing conformational changes


and forming homo-oligomers in the outer mitochondrial membrane (Kroemer et al., 2007). The homo-oligomers then eitheralone or in conjunction with each other, form large protein-permeable openings in the membrane, allowing cytochrome cefflux (Kuwana et al., 2002). The exact nature of the Bax/Bak pores has yet to be elucidated, however parallels have beendrawn between them and the mitochondrial apoptosis induced channel (MAC). The induction of the MAC coincides withBax translocation to the mitochondria membrane, and various functional similarities suggest that they may in fact constitutethe same entity (Kinnally and Antonsson, 2007). Other means by which the Bax/Bak oligomers may induce membrane per-meabilisation is via destabilization of the lipid bilayer (Basanez et al., 2002), or activation of the mitochondrial permeabilitytransition pore (MPTP). Indeed interactions between Bax and components of the MPTP have been observed (Marzo et al.,1998; Shimizu et al., 1999) and it is now thought that Bax may mediate membrane permeabilisation through homo-oligomerpores or the MPTP under different conditions (Pastorino et al., 1999).

It is generally accepted that membrane permeabilisation by Bax and Bak is functionally mediated by another class of Bcl-2family of proteins, the BH3 domain only proteins, which as the name suggests, share only one Bcl-2 homology domain. Mem-bers of this family have two roles, either to bind and activate the multi-domain pro-apoptotic proteins, or to displace anti-apoptotic proteins from the outer mitochondrial membrane. In particular Bid and Bim have been shown to activate Bax andBak directly, whilst Bad and Bik play a more indirect role displacing anti-apoptotic Bcl-2 proteins (Scorrano and Korsmeyer,2003). The main function of anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) is to bind and neutralize Bax and Bak, and so bydisplacing these anti-apoptotic proteins, Bad and Bik indirectly promote membrane permeabilisation.

4.5. Mitochondrial permeability transition pore (MPTP)

Whilst pro-apoptotic Bcl-2 family proteins contribute to cytochrome c release via permeabilisation of the outer mitochon-drial membrane, the MPTP is characterized by disruption of both inner and outer membranes. The MPTP is a large, non-spe-cific pore that spans the inner and outer mitochondrial membranes and is permeable to solutes with a molecular mass of up to1.5 kDa. Whilst there has been some controversy regarding the constituents of the MPTP, consensus is that the multi-proteincomplex centers on the adenine nucleotide translocase (ANT) (Halestrap and Brennerb, 2003). The primary physiologicalfunction of ANT is to exchange mitochondrial matrix ATP for cytosolic ADP, fulfilling an essential role in oxidative metabolism.In an apoptotic scenario however, ANT forms dynamic interactions with the outer membrane voltage dependant anion chan-nel (VDAC) and matrix dwelling Cyclophilin D, to form a pore across the inner-mitochondrial membrane. Formation of theMPTP allows the equilibration of solutes <1.5 kDa across the inner-mitochondrial membrane, an influx of water into the ma-trix, swelling of the mitochondrial matrix and finally ruptures the outer mitochondrial membrane. This in turn permits therelease of cytochrome c and dissipation of the mitochondrial transmembrane potential (Halestrap et al., 2002).

A rise in matrix Ca2+ concentration is the primary trigger for opening of the MPTP, however a number of factors are knownto sensitise the pore to the affects of Ca2+, including adenine nucleotide depletion, increased inorganic phosphate concentra-tions, mitochondrial depolarization, and oxidative stress (Halestrap et al., 2002). Conversely MPTP opening can be inhibitedby ligands of ANT such as bongkrekic acid and ADP, and cyclophilin D ligands such as cyclosporin A (Halestrap and Brennerb,2003; Halestrap et al., 1997a).

Oxidative stress promotes MPTP opening via oxidation of thiol residues within ANT. The ANT contains three matrix facingcysteine residues, and thiol oxidation has been shown to both enhance cyclophilin D binding and inhibit ADP binding to ANT,thus sensitising the MPTP to the effects of Ca2+ (McStay et al., 2002). Menadione, diamide, arsenite, tert-butylhydroperoxide,and the vicinal thiol binding agent phenylarsenoxide (PAO) are all potent activators of the pore through this mechanism(Lenartowicz et al., 1991; Marchetti et al., 1997; Petronilli et al., 1994; Valle et al., 1993; Vieira et al., 2001; Wudarczyket al., 1996). Reactive oxygen species (ROS) generated by mitochondrial dysfunction can also sensitise the cell to MPTP open-ing. Numerous anti-cancer agents such as doxorubicin and arsenic trioxide induce apoptosis by generating ROS that directlyact on the ANT to promote MPTP formation (Oliveira and Wallace, 2006).

Clearly the role of mitochondria in apoptosis is significant, and so have mitochondria become an attractive target for induc-ing cell death. In particular, a number of cancer therapeutics have targeted the mitochondria of tumour cells, as a way of cir-cumventing tumour cell resistance to apoptotic cell death. This is largely because the mitochondria lie downstream from theinitial targets of anti-cancer agents, and are therefore less compromised by mutations accompanying tumour resistance. For acomprehensive review of mitochondrial targeting anti-cancer agents the reader is directed to Don and Hogg (2004) andHuang et al. (2010) earlier in this issue. The purpose of this review however will be to highlight anti-angiogenic therapies thattarget the mitochondria of endothelial cells, induce mitochondrial based apoptosis, and so inhibit tumour growth (Table 2).This group of compounds can be divided into two categories, those that directly target the mitochondria like GSAO and Vita-min E analogues (Fig. 1), and those that initially target other cellular processes but involve the mitochondria downstream.

5. Angiogenic inhibitors targeting the mitochondria

5.1. GSAO and derivatives as mitochondrial poisons

As mentioned, thiol oxidising or alkylating agents are powerful activators of the mitochondrial permeability transition(MPT) that act by modifying one or more of the three unpaired cysteines in the matrix side of ANT (Halestrap et al.,

Fig. 1. Anti-angiogenic therapeutics that directly target the mitochondria. The extrinsic apoptotic pathway is initiated upon ligation of death receptors on theplasma membrane and involves activation of the caspase cascade through caspase 8. The intrinsic apoptotic pathway is characterized by an increase inmitochondrial permeability via formation of the mitochondrial permeability transition pore (MPTP) or Bax/Bak associated pores. The release of cytochromec from the mitochondrial intermembrane space facilitates formation of an apoptosome and activation of caspase 9. Cross talk between the two pathways ismediated by t-Bid which is activated by caspase 8. CAO, a GSAO metabolite, and PENAO promote mitochondrial permeability by binding to the adeninenucleotide translocase (ANT) and inducing the MPTP. Vitamin E analogues a-TOS and a-TEA bind to Complex II in the inner-mitochondrial membrane,leading to displacement of ubiquinone and the generation of reactive oxygen species. YM155, EM-1421 and LY2181308 inhibit survivin, relieving itsinhibitory affects on caspase activation and mitochondrial destabilisation.

Table 2Anti-angiogenic drugs that target the mitochondria.

Anti-angiogenic therapeutic Mitochondrial target

GSAO, PENAO Cross link dithiols on the adenine nucleotide translocase and induce the mitochondrialpermeability transition pore (MPTP)

Vitamin E analogues Displace ubiquinone from Complex II in the inner-mitochondrial membrane leading to productionof reactive oxygen species.

YM155, LY2181308, EM-1421 Inhibit survivin and relieve its inhibitory affects on caspase activation and membranedestabilisation

Paclitaxel Associated with a decrease in Bcl-2, possibly induces the MPTPCollagen derived angiogenic inhibitors

(endostatin, canstatin)Endostatin- associated with a decrease in Bcl-2 and Bcl-XL and induces the MPTP.Canstatin – activates the intrinsic pathway through an unknown mechanism. Possibly throughinhibition of pro-survival signalling

Silibilin Associated with an increase in Bax and decrease in survivin and Mcl-1Neovastat Activates both intrinsic and extrinsic apoptotic pathways through an unknown mechanism


1997b; Lenartowicz et al., 1991; McStay et al., 2002). For this reason, the small trivalent arsenical, PAO is a potent activator ofthe pore (Lenartowicz et al., 1991; McStay et al., 2002). PAO reacts with two thiols in ANT (McStay et al., 2002) forming astable cyclic dithioarsinite in which both sulfur atoms are complexed to arsenic (Adams et al., 1990). Trivalent arsenicalsare selective for closely-spaced dithiols, as the ring structures of trivalent arsenicals and dithiols are markedly more stable


than the non-cyclic products formed with monothiols. There is one cysteine in each of the three loops of ANT that protrudeinto the matrix, Cys57, Cys160 and Cys257. The evidence indicates that PAO cross-links two of these cysteine residues and en-hances cyclophilin D binding while antagonizing ADP binding (McStay et al., 2002). The two effects together greatly sensitisethe trigger site to Ca2+ concentration, which facilitates pore opening.

PAO is lipophilic and is generally toxic for all cultured cells after exposure to nanomolar concentrations for 1 day. Wemade a hydrophilic derivative of PAO, by attaching it to the cysteine thiol of reduced glutathione (GlyCys cGlu), to producea membrane impermeant synthetic tripeptide 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (Dilda and Hogg,2007; Donoghue et al., 2000). We hypothesized that the glutathione pendant is the moiety that mediates GSAO’s entry intocells. Considering that GSAO resembles a glutathione-S-conjugate of aminophenylarsonous acid, we explored the possibilitythat GSAO was metabolized at the cell surface and a product containing the arsenical moiety was transported across theplasma membrane. We demonstrated that the c-glutamyl residue of GSAO is cleaved from the molecule at the cell surfaceby c-glutamyltransferase (cGT; EC 2.3.2.2) to produce 4-(N-(S-cysteinylglycylacetyl) amino) phenylarsonous acid (GCAO).GCAO then enters the cell via an organic ion transporter and is likely further processed by dipeptidases to 4-(N-(S-cysteiny-lacetyl)amino)phenylarsonous acid (CAO) in the cytosol (Dilda et al., 2008). GCAO and CAO, like GSAO or PAO, activated theMPTP and triggered swelling of isolated rat liver mitochondria in a comparable Ca2+-dependent manner (unpublished obser-vations). GCAO and CAO, therefore, have very similar effects on mitochondrial function as GSAO (Fig. 2).

Reaction of the trivalent arsenical moiety of GSAO, GCAO or CAO with two thiol groups in the matrix facing loops of ANTleads to major intracellular changes (Dilda et al., 2008; Don et al., 2003). GSAO treatment induced MPTP activation, caspase-3/7 activation, loss of mitochondrial transmembrane potential, and surface presentation of phosphatidyl serine in bovineaortic endothelial cells cells (BAECs). The binding of GSAO to ANT and loss of mitochondrial transmembrane potential af-fected cellular ATP levels in proliferating BAECs. Cellular levels of superoxide anion (O�2 ) increased linearly with GSAO con-centration in proliferating, but not growth quiescent BAECs. The linear increase in O�2 levels with GSAO treatment isconsistent with a disruption of mitochondrial integrity.

GSAO has little effect on growth quiescent endothelial cells (Don et al., 2003). We have proposed that the selectivity ofthis compound for proliferating endothelial cells is a consequence of the higher mitochondrial Ca2+ levels in proliferatingcells (Don et al., 2003). By investigating the calcium-dependence of the interaction of GSAO with ANT and probing theANT residues that react with GSAO we demonstrated that GSAO, GCAO and CAO triggered formation of the permeability porein rat liver mitochondria in a comparable Ca2+-dependent manner and a biotin-tagged GSAO interacted with Ca2+-repleteANT with higher affinity than Ca2+-depleted ANT (unpublished observations). ANT has three matrix facing cysteine residuesand GSAO reacts with Cys160 and either Cys57 or Cys257 in a calcium dependant manner. The optimal spacing of cysteine thi-olates for reaction with As(III) is 3–4 Å (Adams et al., 1990; Bhattacharjee and Rosen, 1996) whilst all three matrix facing

OATP

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Fig. 2. Mechanism of action of GSAO. GSAO is processed by c-GT at the cell surface and the resulting GCAO is transported across the plasma membrane by anOATP. GCAO is likely further processed to CAO in the cytosol before it reacts with ANT of the inner-mitochondrial membrane. The cytosolic levels of GCAOand CAO are controlled by export from the cell by MRP isoforms 1 and 2.


thiols of ANT are 18–20 Å apart in the crystal structures of the Ca2+-free form of bovine ANT (Nury et al., 2005; Pebay-Pey-roula et al., 2003). This is consistent with the weaker interaction of GSAO with Ca2+-depleted ANT and implies that Ca2+ bind-ing mediates a conformational change in ANT that brings the Cys160 and Cys257/Cys57 sulfur atoms closer together(unpublished observations). In summary, GSAO reacts preferentially with Cys160 and either Cys57 or Cys257 of Ca2+-repleteANT. The higher mitochondrial Ca2+ levels in proliferating cells, therefore, are predicted to favour reaction of CAO withANT which confers selectivity for angiogenic endothelial cells.

GSAO is also a selective inhibitor of proliferating endothelial cells compared to tumour cells (Dilda et al., 2005b). The IC50

for proliferation arrest of bovine or human primary endothelial cells is 10–15 lM in a 3 day proliferation assay, whereas theconcentrations of GSAO that induce proliferation arrest in tumour cells are 3 to >32-fold higher in all lines tested. These cellculture observations are borne out in vivo. Systemic administration of GSAO to immunodeficient mice bearing human pan-creatic BxPC-3 carcinoma tumours reduced tumour vascularity but had no effect on the proliferative index of the tumourcells (Don et al., 2003). To elucidate this mechanism of selectivity we screened a genome-wide set of Saccharomyces cerevisiaedeletion strains to identify those genes that confer resistance to GSAO (Dilda et al., 2005b). A role for the corresponding geneproducts in endothelial and tumour cells was confirmed using cells expressing different drug transporters and by pharma-cological intervention. We found that the multidrug resistance-associated proteins (MRP) 1 and 2 in combination with cel-lular glutathione mediate export of GSAO from mammalian cells. The selectivity of GSAO for endothelial versus tumour cellsis mostly accounted for by differences in MRP1/2 activity and cellular glutathione levels (Dilda et al., 2005b).

The ability of GSAO to selectively kill proliferating, but not growth quiescent, endothelial cells in vitro suggested that itmight be an effective inhibitor of angiogenesis in vivo. Following the demonstration that GSAO inhibited chick chorioallan-toic membrane (CAM) angiogenesis in a concentration-dependent manner we next tested whether GSAO would inhibit tu-mour angiogenesis and tumour growth in mice. The growth of established human and murine primary tumours in immune-compromised and immuno-competent mice was suppressed by administration of GSAO. Several routes of administrationhave been tested: subcutaneous injections, intra-peritoneal injections, drinking water or osmotic pumps implanted underthe skin of the animal. Treatment by GSAO of immuno-compromised mice bearing subcutaneous tumours from human pan-creatic carcinoma or fibroscarcoma as well as immuno-competent mice bearing Lewis lung cancer (LLC) tumours resulted ininhibition of the rate of tumour growth from 45% to 90%. There were no apparent adverse affects of administration of GSAOto either immuno-competent or immune-compromised mice. The average mice weights of the GSAO treatment groups overthe course of the experiments were not significantly different, and there were no macroscopic differences or morphologicalchanges apparent in the heart, lungs, liver, kidneys, and spleen of GSAO-treated mice. Immuno-histochemical analysis of thetumours from the experiment indicated a significant reduction in blood vessel density in the GSAO-treated tumours. Theproliferative indices of the vehicle and GSAO-treated tumours were the same, while there was a significant increase inthe apoptotic indices of GSAO- versus vehicle-treated tumours. The effect of GSAO on tumour growth is consistent with inhi-bition of tumour angiogenesis. GSAO arrested proliferation of endothelial cells in vivo but had no effect on tumour cell pro-liferation (Don et al., 2003).

The ‘‘first in human” trial of GSAO is currently recruiting patients with progressing advanced solid tumours who failedstandard therapies. GSAO is given intravenously to patients in Phase I/IIa clinical trial. To date 18 patients have been enrolledin three cohorts of different dose levels. Dose escalation is ongoing.

We have made a chemically stable analogue of CAO, 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid (PENAO).The cysteine residue in CAO has been replaced with a D-penicillamine residue in PENAO. PENAO does not require processingby c-glutamyl transpeptidase at the cell surface, which was predicted to enhance its efficacy in vitro and in vivo. PENAO accu-mulates in cells much more rapidly than GSAO and CAO, which translates to more potent effects on endothelial and tumourcells in culture. The faster rate of accumulation of PENAO compared to CAO is mostly due to decreased rate of export byMRP1/2 (Fig. 3). PENAO, therefore, is an at least a 20-fold better inhibitor of BAE cell proliferation and cell killing agent thanGSAO (Dilda et al., 2009).

Like GSAO and CAO, PENAO triggered swelling of isolated rat liver mitochondria in a time- and concentration-dependentmanner. Comparison of the time for maximal swelling as a function CAO or PENAO concentration indicates that the two com-pounds are comparable in their effects on mitochondrial integrity (Fig. 3). PENAO is also a �20-fold more effective inhibitorof tumour growth in mice than GSAO. Comparable anti-tumour activity was observed with administration of 0.25 mg/kg/dayPENAO and 5 mg/kg/day GSAO. No systemic toxicity of either PENAO or GSAO was apparent at these doses. Analysis of PE-NAO treated tumours showed a marked reduction in vascular hot spots as well as significant inhibition of BxPC-3 tumour cellproliferation (Dilda et al., 2009).

In an effort to better understand the mechanism of action of GSAO and to refine its efficacy as an anti-cancer drug, wehave designed a second generation of compound in which the position of the arsenical moiety of GSAO is at the ortho,not the para, position on the phenyl ring. The resulting compound was named ortho-GSAO (o-GSAO). We thought thatthe ortho positioning of the arsenical might improve its dithiol reactivity. The electrophilic character of the arsenic atomis predicted to be enhanced through formation of a six-membered intramolecular hydrogen bond between the oxygen ofthe arsenic-bound hydroxyl and the hydrogen of the NH group. o-GSAO is more efficient than GSAO at inducing the mito-chondrial permeability transition. The time for half-maximal swelling of isolated mitochondria was three to five times fasterfor a given concentration of o-GSAO compared with GSAO. Indeed, o-GSAO was almost as efficient as the lipophilic PAO atinducing the permeability transition (Dilda et al., 2005a). Ortho-GSAO, like GSAO, was a selective inhibitor of BAE comparedwith tumour cell proliferation. Ortho-GSAO accumulated in BAE cells at a 300-fold faster rate than GSAO. We demonstrated

OATP

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Fig. 3. Mechanism of action of PENAO. PENAO is transported across the plasma membrane by an OATP and enters the mitochondrial matrix where it interactswith ANT, which triggers proliferation arrest of the cell. The cytosolic levels of PENAO are controlled by export from the cell by MRP isoforms 1 and 2,although its export is not as efficient as for GCAO or CAO.


that the increased accumulation of o-GSAO in BAE cells was due to decreased efflux via MRP1. The growth of establishedhuman primary tumours in immuno-compromised mice was suppressed by administration of o-GSAO. Comparable anti-tu-mour activity was observed with administration of 1 mg/kg/d o-GSAO and 10 mg/kg/d GSAO compared to vehicle treatedmice. Histological exam of tumours excised at the conclusion of treatment showed a significant reduction in blood vesseldensity in the o-GSAO tumours. These findings indicate that o-GSAO is a more potent inhibitor of endothelial cell prolifer-ation and in vivo tumour growth than GSAO. But o-GSAO administration to tumour-bearing mice resulted in side effects notseen with GSAO. These effects can be explained by increased accumulation of o-GSAO in endothelial cells (Dilda et al.,2005a).

5.2. Vitamin E analogues

Vitamin E analogues are part of the mitocan family of compounds first recognized for their selective toxicity to cancercells (Neuzil et al., 2004). This family of compounds is best represented by a-tocopheryl succinate (a-TOS) and its ether ana-logue a-tocopheryloxyacetic acid (a-TEA) which have been shown to suppress cancer growth in several experimental mod-els and more recently in vivo in a number of murine tumour models (see Zhao et al. (2009) for a review). Recently a-TOS anda-TEA were also identified in an anti-angiogenic capacity. The potent anti-cancer effect of these agents in vivo can thus beattributed to the direct inhibition of the tumour itself, and the simultaneous inhibition of the tumour blood supply. LikeGSAO, these Vitamin E analogues selectively trigger apoptosis in proliferating but not growth quiescent endothelial cells.Both a-TOS and a-TEA have been shown in inhibit in vitro models of angiogenesis, whilst a-TOS has been shown to signif-icantly impede tumour vascularisation and tumour growth in vivo (Dong et al., 2007).

The pro-apoptotic affects of a-TOS and a-TEA appear to be functionally mediated by the mitochondria. Indeed endothelialcells deficient in mitochondrial DNA were resistant to a-TOS and a-TEA induced apoptosis. a-TOS and a-TEA treatment wasalso accompanied by disruption of the mitochondrial transmembrane potential and generation of mitochondrial ROS (Donget al., 2007). These events are typical of the intrinsic apoptotic program, and this pathway is likely activated by a-TOSthrough direct action on the mitochondria, and via ceramide formation (Neuzil et al., 2007).

One of the earliest events in cells treated with a-TOS and a-TEA is generation of ROS by the mitochondria. a-TOS and a-TEA have been shown to bind directly to Complex II in the inner-mitochondrial membrane leading to displacement of ubiq-uinones (Fig. 1). The absence of this electron acceptor results in recombination of electrons with molecular oxygen and theformation of ROS (Dong et al., 2007, 2008). As discussed previously ROS are potent activators of the mitochondrial apoptoticpathway, particularly in the context of MPTP formation. a-TOS has been shown to induce MPTP formation in tumour cells(Yamamoto et al., 2000), and it is possible that this is a direct consequence of ROS generation. a-TOS may also influencemitochondrial permeability by shifting the balance of pro- and anti-apoptotic Bcl-2 family proteins (Weber et al., 2003;Yamamoto et al., 2000). Indeed over-expression of Bcl-2 and Bcl-XL protects cells from a-TOS, whilst over-expression of


Bax has the converse affect (Neuzil et al., 2001a,b; Weber et al., 2003). In fact a-TOS may bind and inhibit Bcl-2 and Bcl-XL

directly (Shiau et al., 2006) so destabilising the mitochondrial membrane.Mitochondrial apoptosis induced by a-TOS may also be mediated through ceramide, which is formed upon a-TOS acti-

vation of sphingomyelinase (Weber et al., 2003). Ceramides are thought to negatively influence membrane permeabilityvia translocation of Bax to the mitochondrial membrane (Birbes et al., 2005) and activation of the protease cathepsin D lead-ing to activation of mitochondrial Bid (Heinrich et al., 2004). It has also been suggested that ceramides are involved in theformation of channels in the mitochondrial outer membrane, allowing movement of proteins up to 60 kDa in size, includingcytochrome c (Siskind et al., 2002). Ceramides have also been reported to enhance generation of mitochondrial ROS. (Garcia-Ruiz et al., 1997; Quillet-Mary et al., 1997). By activating sphingomyelinase a-TOS may therefore promote membrane desta-bilisation as well as enhancing ROS production.

The selectivity of the compounds for proliferating vs growth quiescent cells is likely mediated by the formation of ROS.Indeed ROS were only shown to accumulate in proliferating endothelial cells upon treatment with a-TOS and a-TEA, andelimination of ROS by co-exposure to anti-oxidants suppressed the extent of apoptosis. It is possible that this is due tothe upregulation of protective anti-oxidant systems in resistant growth quiescent endothelial cells, such as manganesesuperoxide dismutase (MnSOD). Elevated expression of MnSOD has been found in growth quiescent endothelial cells (Donget al., 2007).

A number of anti-angiogenic compounds induce endothelial cell apoptosis, however to date only a-TOS, a-TEA and GSAOhave displayed selective toxicity for proliferating endothelial cells. This property makes them particularly effective anti-angiogenic therapeutics, as they are likely to affect only newly formed blood vessels, such as those observed in tumourgrowth, and not the pre-existing vasculature.

5.3. Inhibitors of survivin

Survivin is a 17 kDa protein with a dual biological function. It cooperates with a range of mitotic apparatus including cen-trosomes, kinetochores and mitotic spindle microtubules and is an essential mediator of cell division. It is also a member ofthe family of inhibitors of apoptosis proteins (IAP), functioning as a regulator of mitochondrial apoptosis (Altieri, 2008). Sur-vivin is localised in multiple subcellular compartments, however a mitochondrial pool appears to be released into the cytosolupon apoptotic signalling and regulates key aspects of the mitochondrial apoptotic pathway. In the presence of a co-factorsurvivin binds and inhibits caspase 9 (Marusawa et al., 2003). Mitochondrial localised survivin has been shown to sequesterSmac/DIABLO, preventing it from binding caspase inhibitors such as XIAP (Song et al., 2003; Sun et al., 2005). As discussedpreviously Smac/DIABLO promotes apoptosis by interacting with XIAP and preventing its inhibition of caspases. By seques-tering Smac/DIABLO, survivin counteracts these pro-apoptotic actions. Survivin also hinders proteolytic degradation of XIAPin the cytosol (Dohi et al., 2004). A role for survivin as an upstream regulator of mitochondrial membrane permeability hasalso been suggested. A splice variant of survivin has been shown to interact with Bcl-2 and inhibit caspase 3, whilst otherstudies have indicated that a survivin-Aurora kinase B complex may activate and inhibit Bcl-2 and Bax, respectively (Altieri,2008; Vogel et al., 2007; Wang et al., 2002).

Survivin is largely undetectable in normal differentiated tissue but is up regulated in numerous cancers in which it fosterscontinued proliferation and resistance to apoptotic signals (Altieri, 2008). Survivin is also upregulated in endothelial cellsduring the proliferative and remodelling phases of angiogenesis (Harfouche et al., 2002; O’Connor et al., 2000; Papapetrop-oulos et al., 2000). Because survivin expression is almost exclusively limited to these two cell types, it represents an appeal-ing target for anti-tumourigenic and anti-angiogenic therapeutics. Targeting survivin has been shown to induce tumour celldeath and tumour regression in a number of murine tumour models, as well as inducing endothelial cell death and suppress-ing angiogenesis (Blanc-Brude et al., 2003; Xiang et al., 2005). The cell death pathway initiated by survivin targeting was alsoshown to involve release of cytochrome c, Smac/DIABLO and AIF, and loss of the mitochondrial transmembrane potential(Blanc-Brude et al., 2003; Carter et al., 2003).

Currently, only a few direct inhibitors of survivin exist, likely because it is not a cell-surface molecule and its physicalstructure does not lend to potential drug interactions (Altieri, 2008). Those that do exist therefore act at the level of the gen-ome, silencing survivin expression. The transcriptional repressor YM155 has recently completed a Phase I pharmacokineticstudy in patients with advanced solid malignancies and lymphoma (Nakahara et al., 2007; Tolcher et al., 2008), whilst theantisense oligonucleotide LY2181308 is in Phase II trials for the treatment of hepatocellular carcinoma. EM-1421 is anothertranscriptional repressor that has shown promise in the treatment of cervical intraepithelial neoplasia and is now enteringPhase I trials (Chang et al., 2004). Although in the early stages of development, these agents represent another class of agentswith dual activity-directly inhibiting cancer cells and the angiogenic endothelial cells that support them (Fig. 1).

5.4. Paclitaxel

Paclitaxel (Taxol) is potent chemotherapeutic used in the treatment of ovarian, lung and breast malignancies. It functionsprimarily as a microtubule-damaging agent in cancer cells, inducing cell cycle arrest at the metaphase–anaphase transition,and eliciting a mitochondrial based apoptotic response in the cancer cell. Paclitaxel also functions in an anti-angiogeniccapacity, selectively inhibiting endothelial cell proliferation in vivo. The mechanism by which Paclitaxel inhibits endothelialcell proliferation parallels that seen in tumour cells. Disturbance of the microtubule network is followed by G2-M arrest and


cell death characterized by all of the usual hallmarks of the mitochondrial apoptotic pathway. Upon treatment with Paclit-axel, human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1) display a loss inmitochondrial transmembrane potential, significant down regulation of the Bcl-2 protein and permeabilisation of the mito-chondrial membrane. Interestingly an increase in p53 expression was also observed (Pasquier et al., 2004). Paclitaxel hasbeen shown to directly interact with isolated tumour cell mitochondria, initiating the MPTP and so inducing cytochromec release (Andre et al., 2000). It is likely that mitochondrial membrane permeabilisation in endothelial cells is also mediatedby the same mechanism.

Lower concentrations of Paclitaxel can also inhibit endothelial cell proliferation through a slightly different mechanism.Concentrations of between 1 and 10 nmol/L appear to generate a global slowing of the cell cycle, rather than arresting thecell cycle at any one stage. At low concentrations Paclitaxel induces a similar disruption in the mitochondrial transmem-brane potential and changes in Bcl-2 expression, however these changes are transient. It is suggested that at low concentra-tions, Paclitaxel initiates the mitochondrial apoptotic pathway, but stops short of mitochondrial membrane permeablisiationand endothelial cell death. Instead the cell cycle slows, and endothelial cell proliferation is inhibited (Pasquier et al., 2004).Nonetheless, mitochondrial disruption continues to play a significant role in its mechanism of action.

5.5. Collagen derived angiogenic inhibitors

Proteolytic degradation of the ECM is an essential component of early-stage angiogenesis, facilitating the migration ofproliferative endothelial cells. It is fitting then that several proteolytic fragments of ECM proteins have been shown to inhibitendothelial cell proliferation and angiogenesis. Canstatin, endostatin and tumstatin are three proteins derived from the pro-teolysis of collagen in the vascular basement membrane.

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is the most well researched and developed of these, and hasbeen shown to inhibit a plethora of murine and human tumours in mice with negligible toxicity. For a detailed review see(Folkman, 2006b). The mechanism of action of endostatin is truly broad spectrum, as it significantly regulates 12% of theendothelial cell genome including many key angiogenic players. For instance, endostatin downregulates VEGF, HIF-1a, EGFR,and neuropilin, and upregulates Tsp-1 (Abdollahi et al., 2004). Of particular interest to this review however is the role ofendostatin and the other collagen derived angiogenic inhibitors in endothelial cell death. The mitochondrial apoptotic path-way appears to play a key role in this process. Endostatin has been shown to significantly reduce Bcl-2 and Bcl-XL in endo-thelial cells (Dhanabal et al., 1999) and induce endothelial cell death characterized by all of the hallmarks of mitochondrialmediated apoptosis, including loss of mitochondrial transmembrane potential, ATP depletion, cytochrome c release and cas-pase 9 activation. This has recently been attributed to formation of MPTP in endothelial cells (Yuan et al., 2008). Canstatinhas likewise been found to trigger a mitochondrial apoptotic mechanism involving loss of mitochondrial membrane poten-tial and caspase 9 activation (Magnon et al., 2005; Panka and Mier, 2003).

Canstatin, Endostatin and Tumstatin may promote mitochondrial apoptosis by inhibiting pro-survival signalling in theendothelial cell. As discussed previously, integrins are upregulated on the surface of proliferating endothelial cells and facil-itate endothelial cell interaction with the ECM. This in turn initiates a cascade of pro-survival signalling largely mediated bythe mitochondria (Cheresh and Stupack, 2008). Canstatin, Endostatin and Tumstatin all possess integrin binding sites,through which they antagonize endothelial cell:ECM interactions.

Pro-survival signalling by integrins is largely mediated through FAK activation of the PI3K signalling cascade (Cheresh andStupack, 2008). Both Tumstatin and Canstatin have been shown to inhibit FAK/PI3K activation by antagonizing integrin avb3

and integrins avb3 and avb5, respectively (Maeshima et al., 2002; Magnon et al., 2005; Panka and Mier, 2003). Downstream ofPI3K, two signalling molecules enhance mitochondrial membrane stability, Akt and p21-activated kinase 1 (PAK-1) (Man-ning and Cantley, 2007). Akt directly inhibits the pro-apoptotic protein Bad (Datta et al., 1997, 2000) and hinders transcrip-tion of other pro-apoptotic Bcl-2 proteins Bim, Puma and Noxa via forkhead box O (FOXO) and p53 control (Manning andCantley, 2007). Akt also inactivates glycogen synthase kinase 3 (GSK3) relieving its inhibitory affects on the anti-apoptoticBcl-2 family protein Mcl-1 (Maurer et al., 2006). PAK-1 meanwhile inhibits Bad (Schurmann et al., 2000) and translocates Rafto the mitochondria where it suppresses apoptosis by regulating mitochondrial ROS and Ca2+ levels (Alavi et al., 2003; Kuz-netsov et al., 2008). By inhibiting integrin ligation, Canstatin, Endostatin and Tumstatin may reverse pro-survival signallingin the endothelial cell, tipping the balance toward mitochondrial membrane permeabilisation.

Although the mitochondria play an integral role in endothelial cell death mediated by these agents, it is important to notethat the extrinsic pathway also features heavily. Canstatin inhibition of PI3K/Akt signalling has also been associated with adown regulation of the anti-apoptotic protein FLIP, leading to activation of the extrinsic apoptotic pathway in endothelialcells. Activation of the mitochondrial apoptotic pathway may be secondary to, and dependant on this (Panka and Mier,2003).

The use of these collagen derived angiogenic inhibitors therapeutically is limited by the large amount of purifiedrecombinant protein required. Future clinical application is thus dependent on the development of agents that could raisethe level of endogenous expression, or enhance their mobilisation from the ECM. Gene therapy seems to have answered thisneed to some extent (Folkman, 2006b; Magnon et al., 2008). The Folkman laboratory are currently developing modifiedforms of Endostatin to allow up scaled production of the protein, whilst a variant of the protein named Endostar (MedgennBioengineering Yantai, China), has recently been approved by the FDA China for non-small cell cancer of the lung (Ling et al.,2007).


5.6. Silibinin

Silibinin is a flavonolignan from the milk thistle (Silybum marianum) seed that has shown strong anti-cancer and chemopreventative effects in lung (Tyagi et al., 2009), colorectal (Singh et al., 2008) and prostate carcinoma in mice (Singh et al.,2002) and is non-toxic in humans even at very high dosages. It functions primarily as an anti-proliferative and pro-apoptoticagent in tumour cells, but is also involved in an anti-angiogenic capacity. This is associated with a variety of pro-angiogenicpathways, including down regulation of nitric oxide synthase (NOS), COX, HIF-1a, and VEGF (Singh et al., 2008), upregulationof MMP inhibitors TIMP-1 and TIMP-2 and increased expression of Ang-2 and its receptor Tie-2 (Tyagi et al., 2009). Whats-more, silibinin has also been shown to induce endothelial cell death by a mitochondrial dependant mechanism. Silibinintreatment of HUVEC and HMEC cells results in cellular apoptosis characterized by a loss of mitochondrial membrane poten-tial and cytochrome c release. This is likely mediated by a change in availability of Bcl-2 family proteins, as an increase in Baxand decrease in Mcl-1 was also observed (Singh et al., 2005). Interestingly, silibinin also decreased the levels of survivin inendothelial cells, which is thought to inhibit Bax (Vogel et al., 2007). By inhibiting survivin, silibinin may therefore encourageBax associated pores in the mitochondrial membrane.

5.7. Neovastat

Neovastat is a naturally occurring inhibitor of angiogenesis derived from marine cartilage (dogfish) that is in Phase IIIclinical trials for the treatment of lung and kidney cancer, and Phase II clinical trials for multiple myeloma. To date it hasbeen shown to target at least four pro-angiogenic pathways. Neovastat hinders VEGF mediated signalling by interfering withVEGFR-2, inhibits MMP-2, MMP-9 and MMP-12 and stimulates tissue type plasminogen activator leading to accumulation ofangiostatin (Gingras et al., 2003).

Neovastat is also a potent mediator of endothelial cell apoptosis. Neovastat was shown to induce chromatin condensa-tion, DNA fragmentation and caspase activation in BAECs, HUVECs and HMEC-1 cells, but not in U-87 glioblastoma, NIH-3T 3 fibroblast, MCF-7 human breast cancer, SW1353 chondrosarcoma and COS-7 simian kidney cells. Whatsmore, endothe-lial cell death appeared to be mediated by the mitochondrial apoptotic pathway, as Neovastat induced both cytochrome crelease, and caspase 9 activation (Boivin et al., 2002). Caspase 8 was also found to be activated by Neovastat, and it is likelythat some cross talk between the extrinsic and intrinsic (mitochondrial) apoptotic pathways occurs in this mechanism ofaction. Indeed caspase 8 is known to activate the BH3 domain only protein, Bid, setting in motion Bax mediated mitochon-drial membrane permeabilisation and cytochrome c release (Scaffidi et al., 1998). Conversely caspase 9 could also activatecaspase 8 (Engels et al., 2000). Although the exact apoptotic pathway has yet to be elucidated, it is clear that the mitochon-dria play a significant role in Neovastat mediated endothelial cell death.

6. Potential mitochondrial targets for the inhibition of angiogenesis

6.1. Prohibitin

Prohibitins 1 and 2 are two highly conserved proteins ubiquitously expressed in eukaryotic cells. They are known to local-ise within the mitochondria, nucleus and plasma membrane, however the pattern of distribution varies according to the celltype and situation. In the mitochondria, Prohibitins 1 and 2 form hetero-oligomeric complexes in the inner membrane, andlikely function as protein chaperones and/or membrane scaffolds. Alternate roles as regulators of transcription and cell cycleprogression have also been proposed (Merkwirth and Langer, 2009).

Recently Prohibitin 1 was also identified in an angiogenic capacity. In endothelial cells prohibitin localises almost exclu-sively in the mitochondria, and its knock-down was shown to result in mitochondrial dysfunction and endothelial cell senes-cence. In an in vivo model, Prohibitin knock-down inhibited endothelial cell migration and angiogenesis (Schleicher et al.,2008).

It would appear that Prohibitin has an additional biological role providing anti-oxidant defence, and its loss results inmitochondrial dysfunction and generation of ROS. Prohibitin knock-down in endothelial cells resulted in depolarization ofthe mitochondrial membrane and blockage of mitochondrial electron transport at Complex I. Recombination of electronswith molecular oxygen in turn generates ROS, which signal cell cycle arrest and so inhibit endothelial cell proliferationand angiogenesis. Interestingly ROS also mediated cytoskeletal changes impairing endothelial cell migration. Loss of prohib-itin therefore hinders a number of angiogenic processes (Schleicher et al., 2008).

Prohibitin appears to be a potential target for future anti-angiogenic therapies, though it may not be a realistic one. Thedifficulty lies in the ubiquitous expression of prohibitin in eukaryotes, such that the inhibitor need be selective for endothe-lial cells.

Angiogenesis is integral to tumour growth, and its targeting is an effective anti-tumour strategy. A number of anti-angiogenic agents target pro-angiogenic growth factors, cytokines and their receptors; however this can be circumventedby the tumour upon upregulation of other pro-angiogenic factors. An alternative approach has been to directly inhibitendothelial cell function, inducing endothelial cell death and so impeding their participation in new blood vessel formation.The mitochondria are the focal point for a variety of pro-apoptotic signals, and this review has highlighted a number of


anti-angiogenic agents that involve the mitochondria in the execution of endothelial cell death. These agents either interactwith the mitochondria directly, or have other initial targets but involve in the mitochondria downstream.

Acknowledgements

We are grateful to Philip J. Hogg for his critical reading of the manuscript and for the design of Figs. 2 and 3. Danielle Parkis supported by the LH Ainsworth Cancer Res.earch Scholarship, the Beth Yarrow Memorial Scholarship, the Covidien PhDScholarship and an Australian Postgraduate Award. Pierre Dilda is supported by the Cancer Council NSW.

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Mitochondria as targets in angiogenesis inhibition

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