Abstract
In this article, I will be explaining how angiogenic inhibitors are being used to treat cancer. I will discuss what angiogenesis is and how tumors trigger blood vessels to begin to grow into them, provide them with nutrients and oxygen that the tumor requires to survive.
Through analysis of cutting edge research, I will outline the various mechanisms of action drugs can take to prevent angiogenesis in detail, from changing the shapes of protein receptors to infiltrating our DNA. Finally, I will discuss their possible future in the world of medicine as well as some of their dark pasts.
Introduction
More than 1 in 3 people in the UK will develop cancer at some stage during their lifetime; worldwide, it is estimated that 7.8 million people died from cancer in 2008 [1]. So it is of paramount importance that we develop new drugs to help fight cancer. One of the main reasons cancer is so deadly is due to a process called metastasis.
What is metastasis?
Metastasis is when tumor cells penetrate blood or lymphatic vessels then circulate through the intravascular stream, and proliferate at another site [2]. A tumor requires a blood supply in order for some of its cells to break off into it and spread around the body. In the absence of such vascular support, tumors may become necrotic [3]. This means the tumor cells die due to a lack of nutrients and oxygen that would otherwise be provided for by a blood supply, thus preventing metastasis. Once the mass of a tumor has become about the size of a pea [4], its surface area to volume ratio has become too small for it to survive by diffusion alone. It must develop a series of blood vessels by stimulating a process called angiogenesis.
What is angiogenesis?
Tumor angiogenesis is the proliferation of a network of blood vessels which penetrates into the cancerous growth [5]. Angiogenesis occurs naturally, for example in a developing embryo. But in a normal adult human, angiogenesis rarely occurs with endothelial cells only dividing on average, once every 1000 days [6]. Angiogenesis is regulated by both activator and inhibitor molecules [7]. Normally, the inhibitor molecules predominate preventing angiogenesis. The up-regulation of angiogenic factors is not enough – the negative regulators or inhibitors must also be down-regulated for endothelial cell division to take place.
How do tumors stimulate angiogenesis?
The inner cells of a small tumor become stressed when there is a lack of nutrients and oxygen. Because of this they stimulate the external tumor cells to produce angiogenic activators. The two most important angiogenic molecules have been identified as vascular endothelial growth factor (VEGF), a glycoprotein, and basic fibroblast growth factor (bFGF) [5]. Both VEGF and bFGF are over expressed by many types of cancerous tumors [9]. VEGF and bFGF are initially synthesized within the tumor cells and are then secreted into surrounding tissues. The molecules come into contact with and bind to their specific protein receptors on the endothelial cells of the existing capillary network. The endothelial cells now activated, begin to produce matrix metalloproteinases (MMPs). These break down the extra cellular matrix which fill the space between proteins. The extra cellular matrix is made up of proteins and polysaccharides, creating hollow spaces between cells in the tumor. This allows endothelial cells to migrate into the tumor where they begin to divide. The endothelial cells organise themselves to form hollow tubes. The hollow tubes develop into a new vascular system with angiotensin-1, -2, and their receptor Tie-2, stabilizing and maturing the blood vessels [8].
Figure 1: Angiogenesis occurring in a metastatic carcinoma. Note the proliferations of many small capillaries.
Angiogenic Inhibitors
As previously mentioned, the increased production of VEGF and bFGF by the tumours is not enough to trigger angiogenesis. The activator molecules must overcome the dozen naturally-occurring inhibitor proteins that deter angiogenesis. Included in this group of angiogenic inhibitors are angiostatin, endostatin, interferon, thorombospondin, prolaktin and inhibitors of metalloproteinase-1, -2 and -3 [10]. I will be focusing on the inhibitors endostatin, interferon-α, bevacizumab and thalidomide which all have different mechanisms in preventing angiogenesis.
Endostatin is a naturally occurring protein (a 20kDa C-terminal fragment of type XVIII collagen) (11) that inhibits angiogenesis directly by preventing the growth of endothelial cells. It inhibits angiogenesis by binding to αvß1/αvß3 integrin as well as VEGF receptors VEGFR-1 and VEGFR-2 (12). Because the endostatin has bonded to the endothelial cell receptors, it is now blocked so no VEGF can bind to the receptors. This means the endothelial cells are not activated and thus, no longer produce MMPs. Endostatin additionally causes proliferating and migrating endothelial cell to apoptosis [11]. This is useful as existing vascular networks are not harmed, only those that are beginning to grow are inhibited.
Bevacizumab is an artificially manufactured monoclonal antibody. Genetic engineering has produced a protein sequence that is 93% human and 7% murine [13]. The monoclonal antibody is able to irreversibly bind to the VEGF-A produced by tumor cells. It can do this as the molecule is structurally and pharmacologically similar to the natural VEGF antibody [13]. Because the bevacizumab is bonded VEGF-A, the structure of VEGF-A changes. This new conformation is no longer complimentary to its VEGFR-2. VEGF-A does not bind to its receptor. The molecules sent by the tumour cells to the endothelial cells are not received, so the tumor cells cannot communicate with the existing vascular network.
Figure 2: Structure of VEGF
An old drug with a new use is thalidomide. Thalidomide’s original use was as a sedative in the 1950s. However, it was soon taken off the market as it was found to cause birth defects when given to pregnant women. It has recently been found to have anti-angiogenic properties and is currently being used to treat cancer in non pregnant patients. The mechanism of action of thalidomide is currently unknown but new research is currently underway to develop new models of thalidomide’s mechanism. Angiogenesis is highly dependant on cell surface adhesion proteins called integrin, and thalidomide has been shown to dramatically down-regulate these subunits, resulting in a decreased rate of angiogenesis. Integrin, like all proteins, is produced by transcription of DNA into mRNA. It is translated from mRNA into the protein. Thalidomide can intercalate with DNA and ‘slide into’ gaps between DNA nucleotides [14]. Thalidomide only tends to intercalate at guanine sites because its structure is very similar to that of guanine and adenine. The binding of thalidomide in the DNA sequence disrupts the transcription process of DNA to mRNA, so proteins are not correctly formed. For transcription to occur, promoter proteins must bind to the DNA strand at particular points called promoter regions. The most common sequences found in gene promoter sites are TATA and CCAAT. They are 91% prevalent [14]. The remaining 9% of gene promoter sites contain a sequence called a GC box with the code: GGGCGG. This sequence is guanine-rich, so thalidomide has a strong effect. The gene coding for integrin αvß3 is commonly associated with GC boxes [14], indicating a likely high amount of thalidomide integration within this gene. Transcription of the integrin αvß3 gene is reduced, so fewer integrin αvß3 proteins are produced. These integrin αvß3 proteins are important in forming new blood vessels as they allow endothelial cells to ‘stick’ together to produce hollow tubes. By the same process, the transcription of protein tumour necrosis factor alpha (TNFα) is reduced [14]. TNFα is involved with endothelial cell proliferation; without it, new blood vessels will not be able to migrate into the tumor.
The fourth group of angiogenic inhibitors, such as the naturally-occurring protein interferon-alpha (INF-α), interferes with the signalling cascade produced by the tumor cells. INF-α treatment results in the inhibition of VEGF gene expression [15]. Sp1 and Sp3 are transcription factors. Transcription factors bind to promoter regions on genes, activating or deactivating the transcription of the genes accordingly. Several activators of the VEGF promoter act in Sp1 and Sp3 dependent ways [15]. Sp1 and Sp3 are particularly good at activating GC-rich target genes [16]. In a study, it was found that the proximal GC box I (found in the VEGF promoter region -66/-55) was sufficient to confer INF-α responsiveness [15]. Therefore, INF-α inhibits VEGF transcription by inhibiting Sp1 and Sp3 transactivation activity [15]. The mechanism for this is currently unknown, but researchers are testing possible hypothesis, that include the phosphorylation [17] or glycosylation [18] of the Sp1 and Sp3 proteins. Treatment with INF-α decreases VEGF plasma levels and decreases VEGF mRNA concentrations [15]. It should be noted that INF-α treatment does not decrease bFGF plasma levels.
Figure 3: Tumor cells were injected into nude mice. After 10 days, when the tumor was roughly 25 mm2, the mice were treated daily with injections of INF-α. The graph on the left shows significantly decrease tumor growth with treatment compared to without treatment. On the right, the bar graph shows that micro-vessel density is also reduced compared to the control after treatment. [15]
Conclusion
Although all of the aforementioned drugs are currently in use, they all have their respective limitations. Angiogenic drugs do not aim to destroy the tumor [4]. Instead, by limiting their blood supply, they shrink the tumor and prevent it from growing again. The tumor is shrunk to a size in which it can obtain its oxygen and nutrients by diffusion. Long term survival benefits with angiogenic treatment alone has not yet been documented [19], but coupled with chemotherapy or radiation therapy, angiogenic treatments increase survival rates [20] — marginally or substantially? Is there a certain percentage associated?. Paradoxically, destroying vascular networks to tumors coupled with radiation therapy can disrupt the delivery of cytotoxic drugs to tumors [21]. In addition, some angiogenic drugs are very expensive, especially those that use monoclonal antibodies. However, extensive research continues to occur into the potential uses and development of angiogenic drugs. Over time, pharmacologists will improve manufacturing techniques for these drugs, improving their effectiveness and reducing their cost. Angiogenic drugs have also been found to be useful in treating other diseases not associated with cancer, such as macular degeneration and neovascular glaucoma [4]. Angiogenic drugs have exciting potential for both cancer treatment and for use in other diseases. It will be interesting to see how their uses and mechanisms develop in the future.
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