cancer therapy by means of irreversible tumor blood flow ... · we recently showed that ac7700...

Gene Therapy and Molecular Biology Vol 9, page 203

203

Gene Ther Mol Biol Vol 9, 203-216, 2005

Cancer therapy by means of irreversible tumor

blood flow stasis: Starvation tactics against solid

tumors Review Article

Katsuyoshi Hori Department of Vascular Biology, Division of Cancer Control, Institute of Development, Aging and Cancer, Tohoku

University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan

__________________________________________________________________________________

*Correspondence: Katsuyoshi Hori; Department of Vascular Biology, Division of Cancer Control, Institute of Development, Aging and

Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan; Tel: 81-22-717-8532; Fax: 81-22-717-8533; E-mail: k-

[email protected]

Key words: Tumor blood flow, Tumor vessel, Combretastatin A-4, AC7700, Microcirculation, Necrosis

Abbreviations: [18F]fluorodeoxyglucose, (18FDG); blood flow, (BF); combretastatin A-4 phosphate, (CA-4P); combretastatin A-4, (CA-

4); interstitial fluid pressure, (IFP); tumor blood flow, (TBF)

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

Summary

Despite extensive research efforts, effective therapies for advanced cancers have not yet been established, and

development of successful treatment strategies remains the most important task in the field of oncology. Three

significant problems in conventional chemotherapy using cytotoxic drugs require attention: (i) choosing the most

effective drug for individual patients, (ii) delivering a sufficient dose of drug to tumor, and (iii) minimizing severe

side effects of anticancer drugs. Because the cancer cells are themselves the direct target of the drugs, these three

problems cannot be avoided. We recently showed that AC7700 (currently AVE8062) a derivative of combretastatin

A-4, achieved irreversible stasis of tumor blood flow (TBF), thereby causing necrosis of tumor tissue by halting the

supply of nutrients. Such effects were unrelated to cancer type, in that they were found for various solid tumors. In

this review, we summarize our research on AC7700 and tumor vessels and discuss how AC7700 causes stasis of TBF

and why the blood flow does not resume. This technique of attacking tumor by means of blocking TBF largely

avoids the three problems typically encountered in conventional cancer chemotherapy that were mentioned above.

We propose that such starvation tactics constitute a new therapeutic approach to solid tumors, including refractory

cancers which are resistant to conventional cytotoxic drugs and recurrent cancers that have acquired drug

resistance.

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

Hori: Starvation tactics against solid tumors

204

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 µm 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.


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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.

Figure 1. Enlargement of the tumor vascular network. A and B, photographs of the initial stage of tumor [Sato lung carcinoma (SLC)]

vascularization (photographed at a 40x magnification). Arrows indicate the terminal arteriole. T, tumor. A, 0 h; B, 72 h. Bar scale, 500

µm. C and D, overlay tracing of each photograph: the network was photographed at a 400x magnification, individual photographs were

assembled into a montage, a transparent sheet was placed over the photograph, and tumor vessels were traced onto the overlay. The

black vessel is the terminal arteriole; shaded vessels are tumor capillaries; arrows show BF direction. Many tumor capillaries arose from

the end portion of a terminal arteriole and developed centrifugally. The tumor microvascular network from one terminal arteriole makes

up one microcirculation unit. Bar scale, 100 µm. (Reproduced from Hori et al, 1992 with kind permission from Tohoku Journal of

Experimental Medicine (Sendai)).


206

Figure 2. Structure of a tumor microcirculation unit. Left panel, two SLC microtumors (a, b) growing in a transparent chamber

(photographed at a 40x magnification). The red arrows indicate the inlet (feeding) vessels. Bar scale, 500 µm. Right panel, overlay

tracing of a photomontage of the vascular network of tumor (a): the network was photographed at a 400x magnification. A large red

arrow shows the inlet (feeding) vessel; shaded vessels are tumor capillaries; asterisks show outlet (drainage) vessels; small arrows

indicate BF direction. Bar scale, 100 µm. (Reproduced from Hori et al, 1991 with kind permission from Japanese Journal of Cancer

Research).

Figure 3. Blockage of BF in a tumor-feeding vessel by mechanical means. Circulation in a feeding vessel was temporarily interrupted by

using a steel needle (arrows), 100 µm in diameter. A, before interruption; B, after interruption. Note that BF through the tumor vascular

network to which blood was supplied by the feeding vessel stopped completely (B). Bar scale, 250 µm. (Reproduced from Hori et al,

1991 with kind permission from Japanese Journal of Cancer Research).

V. CA-4 and related compounds The chemical structure of CA-4, which has a

trimethoxyphenyl group (the A ring), is shown in Figure

4A. Its structure is similar to that of colchicine (Figure

4B). In fact, CA-4 is known to bind to a colchicine-

binding site (Li and Sham, 2002). Moreover,

podophyllotoxin (Figure 4C), which was shown by Algire

et al, (1954) to cause TBF stasis, also has a

trimethoxyphenyl group and is of interest because it is a

tubulin-polymerizing inhibitor (Kelleher, 1978). Because

CA-4 has a simple structure, many derivatives have been

synthesized; AC7700 (Figure 4D) is one of them.

VI. AC7700 Because the A ring of CA-4 (Figure 4A) is thought

to play an essential role in the pharmacological effects of

the inhibitor, the B ring is the primary target for changes

as CA-4 derivatives are synthesized. The derivative


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Figure 4. Chemical structures of CA-4, colchicine, podophyllotoxin, and AC7700. A, CA-4; B, colchicine; C, podophyllotoxin; D,

AC7700. Note that each compound has the 3,4,5-trimethoxyphenyl group.

AC7700 is produced by exchanging the OH group with an

NH2 group on the B ring of CA-4. This structural change

leads to markedly increased antitumor effects. By means

of binding serinamide to the NH2 group, water solubility

of the compound is increased. In the following text, we

describe the pharmacological effects of AC7700 on tumor

microcirculation.

A. Effects on TBF We studied the effects of AC7700 in a variety of

tumor models, as outlined below.

1. Transplanted subcutaneous tumors AC7700 blocked TBF in different transplanted

subcutaneous tumors (Figure 5) (Hori et al, 1999; Hori,

2003). Figure 5A presents data for LY80 (a subline of

Yoshida sarcoma)(magenta circle), AH109A (a Yoshida

ascites hepatoma)(blue circle), SLC (Sato lung

carcinoma)(green circle), respectively. Cells of these three

tumor types had been transplanted subcutaneously into the

back of Donryu rats. Figure 5B shows data for the human

esophageal tumor line TE8, which was transplanted into

the nude mouse. In all of these cases, TBF decreased

markedly immediately after AC7700 administration, with

almost complete stasis achieved after 30 minutes.

2. Autochthonous primary tumors The proliferation rate of the transplanted tumors

whose TBF data appear in Figure 5A and 5B was rapid:

the volume doubling time was 1.7-2.5 days. However, the

proliferation rate of tumors in cancer patients is markedly

slower than that of tumors transplanted into animals

(Charbit et al, 1971). For this reason, before clinical trials

of AC7700, it was also necessary to determine whether

AC7700 blocks TBF in slowly proliferating tumors. We

therefore administered the carcinogen methylcholanthrene

subcutaneously to produce autochthonous primary tumors

in rats. The volume doubling time of the tumors was 5.4-

55.9 days (12.6 ± 9.0 days). AC7700 produced TBF stasis

in all cases (Figure 5C) (Hori et al, 2001).

3. Tumors growing in internal organs and

lymph node metastases Most clinical cancers for which chemotherapy is

used are cases in which the tumor develops within internal

organs and is accompanied by metastases. Thus, we also

investigated the TBF stasis effects of AC7700 in tumors

within organs and lymph node metastases. Figure 6

illustrates that AC7700 produced TBF stasis in all tumors

regardless of their location (Hori et al, 2002).

4. Microtumors In addition, we studied the effects of AC7700 on

microtumors by means of tumors growing in a rat

transparent chamber. With this intravital microscopic

observation system (Hori et al, 1990), we demonstrated

that AC7700 also produced complete stasis of TBF in

microtumors (Hori et al, 1999). This drug can target

micrometastatic foci dispersed throughout the body.

C. Influences of AC7700 on BF in normal

tissues during TBF occlusion To use AC7700 safely, it is important to know what

BF changes occur in normal tissues during occlusion of

BF to tumor tissue. Therefore, we measured changes in BF

in normal tissues and organs after use of 10 mg/kg

AC7700 (Hori et al, 1999).

BF in the brain cortex decreased by approximately

35%. However, the level returned to normal after 24 h. BF

in the liver decreased about 50%, but it too returned to its

pretreatment level, within 8 h. BF in the kidney cortex

showed almost no changes. The decrease in BF in the bone

marrow was about 80%. However, it returned to 80-90%

of pretreatment level after 24 h. We therefore concluded

that, except for kidney tissue, AC7700 caused a


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Figure 5. TBF stasis effect of

AC7700 on various subcutaneous

tumors. A, magenta circle, LY80, a

subline of Yoshida sarcoma (n = 13);

blue circle, AH109A, a Yoshida

ascites hepatoma (n = 12); green

circle, SLC, a rat lung carcinoma (n

= 18). B, TE8, a human esophageal

cancer (n = 10). C,

methylcholanthrene-induced

fibrosarcoma [10 mg/kg AC7700

group (!, n = 24) vs 0.9% NaCl

group (!, n = 10), P = 0.0082

(repeted measures ANOVA)].

AC7700 solution (10 mg/kg) was

administered i.v. at 0 min. TBF in all

tumors decreased markedly within

30 min. (Reproduced from Hori et al,

1999 with kind permission from

Japanese Journal of Cancer

Research; (Reproduced from Hori et

al, 2001 with kind permission from

Medical Science Monitor).

Figure 6. TBF stasis effect of

AC7700 on tumors growing in

various tissues and organs. A, 0.9%

NaCl group; B, 10 mg/kg AC7700

group. Blue circles, tumor growing

in the kidney [AC7700 group (n =

10) vs 0.9% NaCl group (n = 8), P =

0.0004 (repeated measures

ANOVA)]; brown circles, tumor

growing in the liver [AC7700 group

(n = 10) vs 0.9% NaCl group (n = 8),

P = 0.0007]; black circles, tumor

growing in the muscularis propria of

the stomach [AC7700 group (n = 14)

vs 0.9% NaCl group (n = 8), P =

0.0118]; green circles, metastatic

foci in the cervical lymph node

[AC7700 group (n = 10) vs 0.9%

NaCl group (n = 8), P = 0.0065];

magenta circles, tumor growing in

muscle [AC7700 group (n = 10) vs

0.9% NaCl group (n = 8), P =

0.0243]. AC7700 solution was

administered i.v. at 0 min. TBF in all

tumors decreased markedly within

30 min. (Reproduced from Hori et al,


British Journal of Cancer).

reversible decrease in BF in normal tissues, which is

thought to be a consequence of constriction of arterioles

caused by AC7700 (see below).

D. Changes in glucose metabolism in

various tissues By using [18F]fluorodeoxyglucose (18FDG), Kubota

et al, (2002) investigated changes in metabolic functions

of various tissues (tumor, brain, heart, kidney, intestine,


209

and bone marrow) after administration of 10 mg/kg

AC7700. 18FDG uptake by tumor tissue 1 h after AC7700

administration fell to 1/10th the preadministration level,

with virtually no recovery of uptake after 6 and 24 h. This

finding indicates that metabolic functions of tumor tissue

are lost at a relatively early time. In contrast, metabolic

measures of the brain, kidney, intestine, and bone marrow

at 1, 6, and 24 h after AC7700 administration did not

differ significantly from control levels. 18FDG uptake to the heart increased 2.8-fold 1 h after

AC7700 administration. Uptake returned to normal and

was not significantly different from the control level after

6 and 24 h, which indicates a transient change (Kubota et

al, 2002). Nevertheless, this finding suggests that AC7700

induced a constriction of cardiac vessels and an increase in

the load to cardiac muscle. Therefore, in clinical

situations, caution should be paid to side effects on the

heart and the dose of AC7700 should be determined with

care.

E. Antitumor effects We investigated antitumor effects of AC7700

resulting from TBF stasis as related to growth inhibition,

hematogenous metastases, and survival rate.

1. Growth inhibition Effects of AC7700 on growth of tumors (LY80 and

SLC) and on the body weight of rats are shown in Figure

7 (Hori et al, 1999). LY80 is resistant to various anticancer

drugs in clinical use. We found, however, that AC7700

markedly suppressed proliferation of these LY80 tumors

(Figure 7A) without body weight loss (Figure 7C).

Moreover, symptoms commonly associated with

anticancer drugs, such as anemia, and diarrhea were not

Figure 7. Effects of AC7700 on tumor growth and body weight.!, 10 mg/kg AC7700; !, 0.9% NaCl. A, growth of LY80 tumor; B,

growth of SLC tumor; C, body weight of LY80 tumor-bearing rats; D, body weight of SLC tumor-bearing rats. In LY80 tumor-bearing

rats, AC7700 or NaCl was given i.v. at 8, 11, 14, 17, 20, 23, and 26 days after tumor implantation. In SLC tumor-bearing rats, AC7700

or NaCl was administered i.v. at 10, 13, 16, 19, 22, 25, and 28 days after tumor implantation. In LY80 tumor-bearing rats, differences in

tumor size between the AC7700 group (n = 6) and the NaCl group (n = 6) on days 13-33 after tumor implantation were highly significant

(P < 0.0001). In SLC tumor-bearing rats, the differences in tumor size between the AC7700 group (n = 8) and the NaCl group (n = 8) on

days 15-33 after tumor implantation were highly significant (P < 0.0001). No obvious side effect such as anemia or diarrhea was

observed at the dose used in this experiment. (Reproduced from Hori et al, 1999 with kind permission from Japanese Journal of Cancer

Research).


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seen after administration of AC7700. This finding is

thought to reflect the fact that the target of AC7700 is not

the actively proliferating cells, so that the side effects of

bone marrow suppression and intestinal malfunction do

not occur.

SLC tumor growth inhibition was more marked

(Figure 7B). Although cachexia was induced in SLC

tumor-bearing rats at an early stage of tumor growth, the

overall condition of the rats undergoing AC7700 therapy

was favorable, with the animals showing an increase in

body weight (Figure 7D). In addition, in mice into which

colon 26 adenocarcinoma was transplanted to the cecum

(orthotopically transplanted tumor), tumor growth was

markedly suppressed by AC7700 (Nihei et al, 1999a).

In rats with methylcholanthrene-induced

autochthonous primary tumors, two of five animals

showed a complete cure, with no subsequent tumor

proliferation after a single dose of 10 mg/kg AC7700

(Hori et al, 2001). The effects of anticancer drugs on slow-

growing tumors are usually weak, but the effectiveness of

treatment by means of TBF blockage was unrelated to the

rate of tumor proliferation.

2. Effects on hematogenous metastases Injection of LY80 cells into the tail vein results in

formation of foci of cancer cells in nearly all internal

organs except kidneys. This model of hematogenous

metastases was used to evaluate AC7700 therapy. Five

treatments were given at intervals of 2 days, starting on

day 7 after tumor cell injection. Measurement of the

number and size of tumors in the internal organs after

therapy showed that tumor cell proliferation was

significantly suppressed in all sites examined, including

lung, liver, cardiac muscle, skin, and internal lymph nodes

(mediastinale, celiacum, mesentericum, lumbare)(Hori et

al, 2002). These results support our conclusion that

AC7700 blocks BF to tumors growing in internal organs.

3. Effects on survival The most important criterion for evaluating drug

treatments is survival rate. In general, even when drugs

suppress tumor proliferation, they often have little effect

on survival rate, because both the growth rate of

transplanted tumors and the reproliferation of tumor

remaining after therapy are rapid.

We previously reported that AC7700 significantly

prolonged survival of various tumor-bearing animals (Hori

et al, 1999; Nihei et al, 1999a). In the case of LY80,

survival was prolonged by 7 days, a significant increase.

As mentioned earlier, LY80 is resistant to nearly all

anticancer drugs in clinical use. For this reason, we believe

that the significant increase in survival obtained with

AC7700 is particularly important and again indicates the

effectiveness of the technique of blocking BF to tumor

tissue.

In the case of SLC, the tumors showed strong

coagulation necrosis, and two of eight tumor-bearing rats

were cured completely after scab formation and

dessiduation (Figure 8) (Hori et al, 1999).

Figure 8. Effect of AC7700 on survival rate of tumor-bearing rats. A. Rats received either 0.9% NaCl solution (group I, control) or 10

mg/kg AC7700 (group II) i.v. at 10, 13, 16, 19, 22, 25, and 28 days after SLC tumor implantation. AC7700 significantly prolonged the

survival of tumor-bearing rats [0.9% NaCl group (n = 8) vs 10 mg/kg AC7700 group (n = 8), P = 0.0022 (log rank test)]. B. Two of the

eight rats were cured completely after scab formation (arrow). (Reproduced from Hori et al, 1999 with kind permission from Japanese

Journal of Cancer Research).


211

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 µm 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.


212

Figure 9. Changes in vascular

lumens caused by AC7700. A, SLC

tumor; B, normal subcutis. Arrows

indicate tumor vessels (A) and

capillaries (B). After i.v.

administration of 10 mg/kg AC7700,

the vascular lumens of tumor vessels

constricted or disappeared, and many

vessels had a fine thread-like

appearance. In contrast, vascular

lumens of normal true capillaries

remained open after AC7700

administration. Bar scale, 50 µm.

(Reproduced from Hori and Saito,



C. Changes in tumor interstitial fluid

pressure (IFP) Tumor IFP is an important index for understanding the

movement of water within the tumor interstitium. We

measured AC7700-induced changes in tumor IFP by using

a diffusion chamber method (Hori et al, 1986). IFP within

the tumor fell immediately after AC7700 administration.

When TBF was about zero, the IFP was 40-50% of the

initial value. During the subsequent 6 h, neither TBF nor

IFP returned to normal levels (Hori and Saito, 2003). This

finding leads us to exclude the possibility that the cause of

TBF stasis induced by AC7700 is compression of tumor

vessels brought about by increased tumor IFP.

D. The process leading from TBF stasis to

tumor necrosis From our observations and measurements described

above, we summarized the process by which AC7700

leads to blockage of TBF and necrosis of tumor tissue, as

shown in Figure 10.

Initially, AC7700 produces sustained constriction of

host arterioles, which leads to an increase in vascular

resistance. The continuing high arteriolar vascular

resistance causes a fall in perfusion pressure in the vessels

feeding the tumor downstream and brings the inflow of

blood to the tumor vascular network to a halt. This change

reduces water volume within the tumor interstitium and is

the cause of the fall in tumor IFP.

Because of the complete blockage of TBF, the

lumens of tumor vessels, with their inherently weak

morphology, become extremely narrow or entirely closed.

Even if BF to the tumor subsequently increases, the closed

tumor vessels are highly resistant to reflow. The decrease in TBF and ultimate stoppage produce

a marked reduction in the water volume within the tumor.

Drainage vessels then become filled with erythrocytes,

followed by hemolysis within 2-3 h. The hemolysis leads

to local fibrin embolism within the tumor (data not shown)

and irreversible blockage of TBF.

The complete stoppage of TBF also results in

dysfunction of convection in the tumor interstitium, and a

decrease in the diffusion efficiency within the tumor.

These decreases in turn prevent the supply of nutrients to

the solid tumor and the removal of waste and thereby lead

to the death of tumor cells (Hori and Saito, 2003).

CA-4 phosphate (CA-4P) has been reported to

enhance tumor vascular permeability (Tozer et al, 2001),

and it has been argued that the increase in tumor IFP

brought about by increased vascular permeability is an

important mechanism in CA-4P-induced blockage of TBF

(Tozer et al, 2002). However, blockage of TBF produced

by AC7700, as just described, is unrelated to either

vascular permeability or IFP. The mechanism of action of

these two drugs might be different.


213

Figure 10. Process of AC7700-induced irreversible TBF stasis and tumor necrosis. AC7700 prevents nutrient supply to the tumor, which

is ultimately the cause of necrosis of the solid tumor tissue. See text. (Reproduced from Hori and Saito, 2003with kind permission from


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 " 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


214

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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