A new approach to fighting cancer?

Normal organ growth and development as well as the maintenance of normal homeostasis rely on precisely controlled blood supply by the circulatory system. The circulatory system assures adequate and specific needs of oxygen and nutrient supplies to each organ. This need is one of the reasons that the circulatory system is the earliest organ system to develop during the development of the fetus. Tumor development and growth can be viewed as uncontrolled tissue growth. As normal organ development and growth relies on the blood supply, tumor growth replies on blood supply by the blood vessels (1–3). It has been known that a growing tumor secretes factors that induce blood vessel growth (i.e., neovascularization) to support its own growth and survival (3). During the last two decades, factors regulating blood vessel formation have been searched for by a number of laboratories. A primary cell type of the blood vessels, especially microvessels in a tumor, is an endothelial cell. Therefore, focus of the search has been to discover factors that specifically control endothelial cell proliferation. During the last several years, a number of molecular biological tools as well as useful cell culture and other in vitro systems to test biological activities of the putative angiogenic and anti-angiogenic factors have become available. These tools have led to the discovery of a number of factors that either stimulate (collectively called angiogenic factors) or inhibit (collectively called antiangiogenic factors) the proliferation of endothelial cells (Table 1).

Table 1.

A list of angiogenic and antiangiogenic factors.

Angiogenic factors	Antiangiogenic factors
Basic fibroblast growth factor	TGF-β
Vascular endothelial growth factor	Lymphotoxin
Scatter factor (HGF)	Inteferon-γ
Transforming growth factor-α	Platelet factor-4 fragment
Epidermal growth factor	Fumagillin (AGM1470)
B61	Shark cartilage extract
IL8	16-kDa Prolactin fragment
Angiopoietin-1	Angiopoietin-2
Angiogenin	Thrombospondin
	Angiostatin (proteolytic fragment of plasminogen)
	Endostatin (proteolytic fragment of collagen XVIII)
	Interferon-γ inducible protein 10 (IP-10)
	2-Methoxyestradiol (endogenous estrogen)
	Genistein

Negative regulators can be used directly to inhibit the proliferation of endothelial cells and, as a consequence, to inhibit the formation of blood vessels. The other approach would be to develop antagonists for the positive regulators of endothelial cell proliferation and to use them to inhibit the blood vessel formation. This rational therapeutical approach, called angiostatic therapy, has been recently shown to be promising based on the discovery of several strong and specific endogenous inhibitory polypeptides for the proliferation of endothelial cells. These factors are angiostatin (4) and endostatin (5).

The discovery of angiostatin was prompted by a long-standing clinical “wisdom” of surgeons. It has been known for a long time that the removal of certain tumors can be followed by the rapid appearance of metastases. One of the explanations of this clinically observed phenomenon is that a primary tumor, while capable of inducing angiogenesis in its own vascular bed, can inhibit angiogenesis in the vascular bed of a metastasis. Based on this hypothesis, O’Reilly et al. (4) attempted biochemical purification of a potent endogenous inhibitor of angiogenesis from the serum and urine of mice carrying a primary tumor and successfully purified such a factor called angiostatin. Purified angiostatin inhibited angiogenesis in both in vitro and in vivo assay systems and blocked growth of metastases. Sequence analysis of angiostatin revealed that it is a proteolytic fragment of plasminogen. The inhibitory activity of angiostatin is specific to this proteolytic fragment because the intact plasminogen lacked this activity. More recently, another endogenous inhibitor of angiogenesis was discovered based on the same principle by the same group. This factor, named endostatin, is also a proteolytic fragment of another protein, collagen XVIII (5). Endostatin was shown to be a more potent inhibitory factor than angiostatin. Systemic application of recombinant endostatin was capable of inhibiting angiogenesis as well as blocking growth of several primary tumors. Furthermore, experimental animals do not seem to develop drug resistance to the endostatin therapy, a critical aspect for a long-term therapeutical strategy (6).

The first step toward using such inhibitors for human therapy is to determine the most effective delivery method. Previously, systemic injection of angiostatin in the experimental animal model was tested, and it yielded a promising but, yet, not a perfect result (7). Tumor regression by repeated systemic injection of angiostatin was able to reduce the size of tumor mass but not to the extent that one would have hoped for. The other problem was that angiostatin used in the study was a purified protein from serum. It would be labor intensive and very expensive to use purified protein from serum for human therapy. Furthermore, purified protein from human serum may pose some potentially dangerous contamination problems. One of the solutions to these problems is to use gene delivery system to express angiostatin “gene” in tumor.

The current paper by Griscelli et al. (8) in this issue describes such a therapeutical strategy. They delivered a portion of angiostatin gene, a domain known to function as an inhibitor for endothelial cell proliferation, by adenovirus-mediated gene delivery system. They showed that this gene delivery system can suppress the proliferation of endothelial cells in vitro as well as in vivo and that the treatment resulted in the regression of tumor mass. They also have shown that this inhibitory effect results from the blockage of endothelial cell proliferation associated with a mitosis arrest. This achievement is a significant step toward specific and cost effective therapy to cure cancer. However, it is certainly only a starting point for better therapeutical approaches. Some of the obvious improvements can be as follows:

1. Specific and more effective delivery methods.

It would be more effective if the delivery of genetic materials can be specifically targeted to the vascular bed of a tumor. This gene-delivery can be achieved, perhaps, by using endothelial specific regulatory elements to drive the expression and/or by engineering the virus itself so that it infects or survives/replicates only in endothelial cells in a tumor. It is also worthwhile to test other gene delivery systems such as retrovirus-mediated gene transfer (9, 10), direct DNA injection (11), and liposome-mediated gene transfer (12, 13) methods for their efficacy.

2. Universality of the therapy.

Both angiostatin and endostatin treatment seem to work with many varieties of tumors. However, more studies are necessary to test whether growth of “all” kinds of tumors can be effectively suppressed by this therapy. It is also important to determine whether there are any contributions by age-related factors. Is the angiostatin/endostatin therapy effective with cancer patients in any age group? Is the angiostatin/endostatin therapy “side-effect-free” with cancer patients in any age-group?

Now, it seems clear that angiostatic therapy is promising as one form of cancer therapy. Based on the discovery of a still-growing number of factors that regulate angiogenesis during the last several years, it would be interesting to see whether other factors shown in Table 1 may serve as alternative or more effective candidates for drug development. The drug development could be targeted to the factors themselves, their receptors, their cofactors, and/or downstream signaling pathways. Furthermore, recent studies have shown that extracellular matrix molecules such as integrins (14) and metalloproteinases (15) also could serve as targets for such drug development efforts. Alternatively, toxins can be targeted specifically to tumor vessels to inhibit angiogenesis and tumor growth (16, 17). Further studies of basic molecular mechanisms of blood vessel formation will most likely lead to the development of novel and more effective therapeutical approaches in the near future. Certainly, more extensive studies of the mechanisms of blood vessel formation as well as molecular and genetic studies of tumor cells themselves are necessary to reach our long-standing goal of fighting cancer. But, hopefully, it will not be too long before we can overcome our fight against cancer.