I. Introduction

Cancer is a leading cause of death in many industrialized nations. Despite development of many treatments, effective approaches for all but early-stage cancers have remained illusive. Chemotherapy is typically used for advanced cancers accompanied by metastasis. The ultimate goal in chemotherapy is elimination of all cancer cells from the body by means of anticancer agents; thus, the target of the anticancer drugs has been the cancer cells themselves. Unfortunately, substances with strong cytocidal effects on cancer cells show similar effects on other, rapidly dividing normal cells, such as hematopoietic cells and mucous membrane cells of the alimentary canal, which results in severe side effects. Some 60 years have elapsed since the first use of an anticancer agent nitrogen mustard (Gilman, 1963), but a substance with selective toxicity for cancer cells has yet to be found.

In recent years, interest has grown in therapeutic methods that target tumor vessels rather than tumor cells themselves. These approaches can be divided into two groups: one that suppresses tumor angiogenesis and one that focuses on blocking the flow of blood in the vessel that provides nutrition to the tumor. The former approach is based on the hypothesis offered by Folkman, (1971) that the tumor can forced into a dormant state if construction of the new vascular lifeline needed for tumor cell proliferation can be prevented. Thus far, many angiogenesis-preventing substances have been screened, several of which are being used in clinical trials. Recently the anti-VEGF antibody bevacizumab (Avastin) has been approved (Ferrara, 2004). Because many reviews of antiangiogenic therapy are available (Kerbel, 2000; Liekens et al, 2001; Eskens, 2004; Cao, 2004), these drugs are not discussed here.

Studies of the latter approach blocking the flow of blood began in the late 1940s, when podophyllotoxin (isolated from the rhizome of Podophyllum peltatum) was found to induce extensive necrosis within tumors (Kelly and Hartwell, 1954). In 1954, Algire et al demonstrated that this necrosis was caused by the blocking of tumor blood flow (TBF) by podophyllotoxin. Unfortunately, this toxin shows significant toxicity and has not been used clinically.

About 30 years later, in 1983, Denekamp et al, performed experiments in which the blood flow (BF) in vessels feeding the tumor was mechanically blocked; they found a linear relationship between the duration of blockage of tumor perfusion and the suppression of tumor proliferation. Thereafter, hydralazine (Chaplin, 1989) and flavone acetic acid (Hill et al, 1989; Zwi et al, 1989) were found to greatly reduce TBF; many studies have examined the effects of these substances in combination with anticancer agents, radiotherapy, and hyperthermia (Chaplin et al, 1989; Horseman et al, 1991; Sakaguchi et al, 1992; Kozin et al, 1994). However, in animal experiments, the decrease in TBF induced by these substances was accompanied by markedly reduced mean arterial blood pressure (Stone et al, 1992).

In 1989, Pettit et al, discovered a new substance combretastatin A-4 (CA-4). CA-4, isolated from the bark of the South African bush willow Combretum caffrum, is a compound that inhibits tubulin polymerization and in vivo shows strong suppression of tumor perfusion (Dark et al, 1997). Many studies have since described the effects of CA-4 against various solid tumors (Horsman et al, 1998; Beauregard et al, 1998; Tozer et al, 1999; Landuyt et al, 2000; Malcontenti-Wilson et al, 2001), and many CA-4 derivatives have been synthesized (Pettit et al, 1995; Hatanaka et al, 1998; Ohsumi et al, 1998; Nam, 2003). A water-soluble derivative of CA-4 with markedly enhanced antitumor effects known as AC7700 (currently AVE8062) was developed (Hatanaka et al, 1998; Ohsumi et al, 1998). We studied the effects of AC7700 on tumor microcirculation and showed that its antitumor effects are due to irreversible blockage of TBF (Hori et al, 1999) and that it is effective in various solid tumors (Hori et al, 1999, 2001, 2002; Nihei et al, 1999a, b). Moreover, in recent studies we clarified the microcirculatory mechanism of this irreversible TBF blockage (Hori and Saito, 2003; Hori, 2003; Hori and Saito, 2004).

In the present review, we summarize our studies on AC7700-induced irreversible TBF stasis and the consequences of this effect for treatment of solid tumors. For a proper understanding of the hemodynamics of TBF stasis, we first discuss the formation of the tumor vascular network and the unusual structural and functional characteristics of tumor vessels. We then focus on the effects of AC7700 on tumor microcirculation. Finally, we discuss how this drug disrupts the function of the tumor microcirculation, which leads to necrosis of tumor tissue.

II. Formation of the tumor vascular network and blood supply

On the basis of intravital microscopic observations, we concluded that tumor angiogenesis is particularly active at the end of host terminal arterioles, which lead into true capillaries (Hori et al, 1990). Tumor blood vessels develop centrifugally from that region and form a larger vascular network (Figure 1) (Hori et al, 1992). The tumor and tumor vessels appear to develop at the trunk of terminal arterioles. In the microvascular system of normal tissue, it is possible to distinguish among arterioles, true capillaries, postcapillary venules, and venules (Wiedeman, 1984). The vascular system of tumor tissue, however, shows marked morphological changes, and it becomes impossible to identify anything but arterioles. In this arteriolar system, although the vessel diameter and framework become enlarged, few changes occur in the pattern of vessel distribution (Hori et al, 1991) and the constrictor function (Hori et al, 1993).

Figure 1 obtained by intravital microscopic observation illustrates that the blood supply to the tumor is clearly governed by a terminal arteriole from the start of formation of the tumor vascular network.

III. Structure and function of a tumor microcirculation unit

By randomly selecting a vessel within the tumor vascular network and following it upstream, one arrives at a single blood vessel (a modified terminal arteriole) that is the feeding vessel of the tumor. By following it downstream, one arrives at drainage vessels. TBF within a network perfused by a single tumor-feeding vessel typically converges into flow in a single or several drainage vessels (Figure 2). The microvascular network that includes all vessels from this feeding vessel to the drainage vessel is the smallest architectural unit of circulatory function and can be considered the microcirculation unit. Chambers and Zweifach, (1944) discovered that the normal vascular bed is composed of microcirculation units and that the size and structure of a microcirculation unit in normal tissue are extremely stable. In contrast, both size and structure of a microcirculation unit in tumor tissue change with alterations in tumor perfusion. A microvascular network within a tumor with a diameter of 500 mm in an early stage is made up of only one microcirculation unit. As the tumor and vascular network develop, the pressure within the feeding vessel increases (Hori et al, 1991), so the size of the microcirculation unit increases (Figure 1). As the tumor grows, its vascular network becomes composed of multiple microcirculation units (Hori et al, 1991).

On the basis of anatomical position and function, we classified vessels within a tumor microcirculation unit into three types (Hori, 2003): (i) tumor-feeding vessels that supply blood to the tumor vascular network, (ii) tumor capillaries that play a central role in exchange of substances within the vascular network, and (iii) vessels that allow removal of blood from the tumor vascular network, i.e., drainage vessels. In Section VII-B, we discuss how these different vessels respond to AC7700.

IV. Blockage of a tumor-feeding vessel by mechanical means

As previously mentioned, BF in the tumor vascular network is governed by feeding arterioles beginning early in the formation of the network. Thus, if the BF of the feeding vessels is blocked, BF in the microcirculation unit as a whole stops. Figure 3 shows the effect of mechanical blockage of BF in tumor-feeding vessels (Hori et al, 1991). Flow of blood to the whole region of perfusion stopped. As will be discussed in Section VII, BF of feeding vessels can also be selectively blocked by using AC7700.

Ohno et al, (2002) also reported that AC7700 significantly prolonged survival of rats bearing Yoshida ascites hepatoma AH130 transplanted to the liver (orthotopically transplanted tumors). Moreover, a markedly prolonged survival rate was found in colon 38 tumor-bearing mice (Nihei et al, 1999a), which suggests that no species differences exist with regard to the effectiveness of AC7700.

VII. Microvascular mechanism of TBF stasis and induction of necrosis

In Section VI, we showed that AC7700 blocks TBF and induces necrosis of solid tumors. In this section, we address questions about the microcirculatory effects of AC7700.

A. Does AC7700 act directly on tumor vessels?

To clarify the mechanism by which AC7700 induces irreversible TBF stasis, it is essential to know the effects of this drug on microcirculation. The first question is therefore whether AC7700 acts directly on tumor vessels to bring about stasis or whether it acts on the host vascular response and thus has indirect effects on tumor vessels.

In these investigations, we dropped a small quantity of AC7700 directly on the region at which BF could be measured and followed BF changes in these regions after topical application of the drug. We found markedly greater sensitivity of normal vessels to AC7700 compared with the sensitivity of tumor vessels (Hori and Saito, 2003). Even with application of AC7700 to tumor vessels at a concentration greater than that thought likely to reach the tumor after intravenous administration, TBF did not change to a great extent. However, when AC7700 was given intravenously to the same animals, complete stasis of TBF occurred within 30 min at sites that showed no changes after topical application (Hori, 2003).

This finding strongly suggests that the main site of AC7700 action may not be the tumor vessels themselves. If AC7700 does not act directly on tumor vessels, how does it induce blockage of TBF, and why do tumor vessels occluded by AC7700 show little tendency to resume normal BF?

B. Vessel reaction to AC7700

To address these questions, we used a transparent chamber for direct observation of responses of host arterioles and tumor vessels to AC7700. The results reported below are based on experiments with 36 rats.

1. Host arterioles

The arteriolar system in subcutaneous tissue, as in other organs, shows a hierarchical structure, with bifurcation of arterioles and eventually, after several branchings, arrival at terminal arterioles (Hori et al, 1990). AC7700 produces constriction but not blockage of all arterioles of this hierarchy, which leads to an increase in vascular resistance (Hori and Saito, 2003) and thus an increase in systemic blood pressure (Hori et al, 1999).

2. Tumor-feeding vessels

As a result of the increased vascular resistance in host arterioles caused by AC7700, vessels feeding the tumor downstream show BF stasis (Hori and Saito, 2003). As discussed in Section II, one feeding vessel provides all blood to the tumor microcirculation unit and tumor vascular network usually consists of many microcirculation units supplied blood by each feeding vessel. BF stasis in feeding vessels thus leads to stasis of BF in the entire tumor vascular network.

3. Tumor capillaries

Tumor capillaries are usually composed of one layer of endothelial cells (Papadimitriou and Woods, 1975; Kornerding et al, 1989). Even when many pericytes and smooth muscle cells are present around the tumor vessels, the relation between the tumor endothelial cells and pericytes or smooth muscle cells is weak (Morikawa et al, 2000; Baluk et al, 2003; Inai et al, 2004). Therefore, tumor vessels cannot maintain a constant morphology; rather, lumens show passive enlargement (mechanical distension) when TBF increases (Suzuki et al, 1984) and passive reduction when TBF decreases (Hori et al, 1993). Even in vessels that had initially been maintained in an enlarged state because of abundant BF, AC7700 produces occlusion of BF or stricture or complete closure of the vascular lumen, such that the vessel often becomes thread-like (Figure 9A) (Hori and Saito, 2003). Stricture or complete closure of the lumen makes the tumor vessel highly resistant to subsequent reflow.

In contrast, the morphology of the vast majority of normal blood vessels was largely unaffected by changes in BF. The lumens of true capillaries 10-15 mm in diameter, unlike the lumens of tumor vessels, remained unchanged after AC7700 administration (Figure 9B). The stability of the morphology of normal capillaries is due to the support of the continuous basement membrane and pericytes (Simionescu and Simionescu, 1984; Baluk et al, 2003; Inai et al, 2004). The normal vascular lumen was maintained even with complete BF stasis after the death of the animal. Because of this structural stability, BF in normal vessels recovers within a short time, even with some decrease in BF as a result of AC7700.

4. Drainage

The most dramatic change seen after AC7700 administration occurs in the drainage vessels of the tumor vascular network. Such vessels have a sinusoidal structure, and most are dilated. BF in the drainage vessels is normally slow, and AC7700 causes further slowing. Thirty minutes after AC7700 administration, many erythrocytes stagnated within the drainage vessels, and complete stasis of BF ensued. Erythrocytes trapped within the lumen lysed after 2-3 h of BF stasis, and complete embolization of the lumens of the tumor vessels occurred (Hori and Saito, 2003). The relationship between lysis and embolization remains unclear at present.

E. Verification of the hemodynamic mechanism by means of epinephrine

If the hemodynamic mechanism for irreversible TBF stasis induced by AC7700, as discussed above, is a general one, drugs other than AC7700 that produce persistent blockage of BF in tumor-feeding vessels should cause irreversible TBF stasis, similar to that caused by AC7700. We tested this hypothesis by using epinephrine (Hori and Saito, 2004). We used epinephrine because its site of action for increasing arteriolar vascular resistance is extremely similar to that of AC7700 (Hori et al, 1993b; Hori and Saito, 2003), although the duration of the effect

is notably different. The effects of AC7700 persist for 2-3 h after administration, whereas those of epinephrine disappear when the drug is no longer given. We therefore hypothesized that prolonging the administration of epinephrine for 2-3 h could induce tumor necrotic effects similar to those of AC7700.

We found that TBF returned to normal immediately after 0.3 mg/ml epinephrine administration when the drug was applied for only 30 min (at a rate of 0.015 ml/min). After a 60-min administration, TBF recovered, but not to the original level. After a 120-min administration, however, TBF did not recover the BF stoppage was irreversible like the case of AC7700. Moreover, extensive necrosis within the tumor was found, as predicted (Hori and Saito, 2004).

We conclude that the hemodynamic mechanisms by which AC7700 and epinephrine stop TBF are extremely similar. Although it is still uncertain why AC7700 causes stricture of host arterioles, it is likely that the site of action of AC7700 is similar to that of epinephrine, i.e., vascular smooth muscle. Further study is required to determine whether specific receptors for AC7700 exist on smooth muscle and whether the epinephrine a receptor is involved in the increased vascular resistance induced by AC7700.

VIII. Evaluation of the therapeutic effect

The degree of reduction in tumor size is generally thought to be important for evaluation of cancer treatment. Our own research has shown that tumor size is markedly reduced when tumor cells are destroyed but that tumor vessels still function. In contrast, however, the degree of tumor size reduction is relatively small when both tumor cells and tumor vessels are destroyed, as is the case with AC7700 (Hori et al, 2003). To obtain prominent reductions in tumor size, the destroyed tumor cells must be moved to outside of the tumor region. For that purpose, scavenger cells (neutrophilic leukocytes and macrophages) must enter the tumor mass and process the tumor cells destroyed by the treatment. Because blockage of BF to the tumor prevents the arrival of scavenger cells, tumor debris that follows necrosis remains at the site of the once-active tumor. For this reason, therapy with AC7700 does not reduce tumor size to a great degree, even though the tumor itself has been destroyed (Hori et al, 2003).

In light of these findings, we conclude one cannot use tumor size reduction as the basis of the evaluation of the effectiveness of drugs such as AC7700 against solid tumors. For a more precise evaluation, greater attention should be paid to intratumor hemodynamics (Gee et al, 2001; Anderson et al, 2003; Stevenson et al, 2003; Thoeny et al, 2005) and changes in tumor markers (Bocci et al, 2004).

IX. Conclusion

Three significant problems in conventional cancer chemotherapy must be addressed. The first concerns drug sensitivity. Because cancer cells in each patient have diverse properties, it is essential to select drugs that have appropriate therapeutic effects. Recent research has focused on drug sensitivity at the genetic level (Zembutsu et al, 2002), but clinical application of the research will take time. The second problem concerns drug delivery. Delivery of anticancer drugs to tumor tissue depends greatly on TBF (Suzuki et al, 1981, 1984; Jain and Ward-Hartley, 1984). However, TBF decreases with increasing tumor volume (Gullino and Grantham, 1961; Vaupel et al, 1987; Hori et al, 1993a). To enhance delivery of anticancer drugs to tumor tissue, we must increase TBF when the drugs are administered (Suzuki et al, 1981, 1984; Sato et al, 1995). The third problem pertains to side effects. Substances thus far screened as candidate anticancer drugs have been those with cytocidal effects on rapidly dividing cells; therefore, side effects on rapidly proliferating normal tissues are, in principle, inevitable.

As discussed above, treatments that focus on blockage of TBF contrast sharply with anticancer drug therapies. By causing selective dysfunction of tumor vessels, we can attack solid tumors through starvation tactics rather than a direct assault on the cancer cells themselves. Therefore, the three problems in cancer chemotherapy that were just mentioned can be largely avoided. Starvation may become an effective strategy for all solid tumors, including refractory cancers, because the therapeutic effect is independent of tumor location and type.

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Review Article

Received: 5 May 2005; Accepted: 14 July 2005; electronically published: September 2005

References