A novel combretastatin A-4 derivative, AC7700, strongly stanches tumour blood flow and inhibits growth of tumours developing in various tissues and organs

Abstract

In a previous study, we used subcutaneous LY80 tumours (a subline of Yoshida sarcoma), Sato lung carcinoma, and methylcholanthrene-induced primary tumours, to demonstrate that a novel water-soluble combretastatin A-4 derivative, AC7700, abruptly and irreversibly stopped tumour blood flow. As a result of this interrupted supply of nutrients, extensive necrosis was induced within the tumour. In the present study, we investigated whether AC7700 acts in the same way against solid tumours growing in the liver, stomach, kidney, muscle, and lymph nodes. Tumour blood flow and the change in tumour blood flow induced by AC7700 were measured by the hydrogen clearance method. In a model of cancer chemotherapy against metastases, LY80 cells (2x10(6)) were injected into the lateral tail vein, and AC7700 at 10 mg x kg(-1) was injected i.v. five times at intervals of 2 days, starting on day 7 after tumour cell injection. The number and size of tumours were compared with those in the control group. The change in tumour blood flow and the therapeutic effect of AC7700 on microtumours were observed directly by using Sato lung carcinoma implanted in a rat transparent chamber. AC7700 caused a marked decrease in the tumour blood flow of all LY80 tumours developing in various tissues and organs and growth of all tumours including lymph node metastases and microtumours was inhibited. In every tumour, tumour blood flow began to decrease immediately after AC7700 administration and reached a minimum at approximately 30 min after injection. In many tumour capillaries, blood flow completely stopped within 3 min after AC7700 administration. These results demonstrate that AC7700 is effective for tumours growing in various tissues and organs and for metastases. We conclude that tumour blood flow stanching induced by AC7700 may become an effective therapeutic strategy for all cancers, including refractory cancers because the therapeutic effect is independent of tumour site and specific type of cancer.

Figure 1

Tumour blood flow changes caused by AC7700 in tumours growing in various tissues and organs. AC7700 at 10 mg kg⁻¹ or 0.9% NaCl solution was infused via the lateral tail vein at the rate of 0.15 ml min⁻¹ by an infusion pump at 0 min. The TBF significantly decreased in all tumours after AC7700 administration compared with the control group. The TBF of tumours in the liver, muscle, and lymph node was completely stopped 30–60 min after AC7700 administration. (A) tumour growing in the liver (open circle, 0.9% NaCl solution (n=8) vs solid circle, 10 mg kg⁻¹ AC7700 (n=10), P=0.0007); (B) tumour growing in the muscularis propria of the stomach (open circle, 0.9% NaCl solution (n=8) vs solid circle, 10 mg kg⁻¹ AC7700 (n=14), P=0.0118); (C) tumour growing in the kidney (open circle, 0.9% NaCl solution (n=8) vs solid circle, 10 mg kg⁻¹ AC7700 (n=10), P=0.0004); (D) tumour growing in the muscle (open circle, 0.9% NaCl solution (n=8) vs solid circle, 10 mg kg⁻¹ AC7700 (n=10), P=0.0243); (E) metastatic foci in the cervical lymph node (open circle, 0.9% NaCl solution (n=8) vs solid circle, 10 mg kg⁻¹ AC7700 (n=10), P=0.0065).

Figure 2

Representative tracings of MABP and the hydrogen gas clearance curve. Upward arrow, start of 9% H₂ inhalation; N, clearance curve in kidney cortex (normal tissue); T, clearance curve in tumour growing in the kidney; downward arrow, starting point of i.v. injection of 10 mg kg⁻¹ ml⁻¹ AC7700; the bold line at the bottom, chart speed 3 cm min⁻¹; the thin line at the bottom, chart speed 60 mm min⁻¹. Tissue blood flow was calculated from the half-life of H₂ washout. Before AC7700 administration, tissue blood flow in the normal kidney cortex and tumour was 157.5 and 30.9 ml min⁻¹ 100 g⁻¹, respectively. After AC7700 was administered i.v., MABP transiently decreased from 113 to 80 mmHg and then immediately increased to 157 mmHg. Under AC7700-induced hypertension, although tissue blood flow in the kidney cortex showed almost no change (150.7 ml min⁻¹ 100 g⁻¹), the TBF decreased to 13.8 ml min⁻¹ 100 g⁻¹.

Figure 3

Photomicrograph of a measured region in a renal tumour. A typical histological feature is present: glomerulus survives within the tumour. H&E-stained tissue section. Original magnification, ×400. Bar, 50 μm.

Figure 4

Antitumour effect of AC7700 against tumours growing in various tissues. The left side in each panel shows results for the group given 0.9% NaCl (eight rats); the right side in each panel shows results for the group treated with 10 mg kg⁻¹ AC7700 (eight rats). The sum of tumours that appeared in rats in each group is shown in each panel. (A) tumours in the lung; (B) tumours in the liver; (C) tumours in the myocardium; (D) tumours in the subcutis; (E) lymph node metastases (mediastinale, coeliacum, mesentericum, lumbare); (F) changes in body weight of tumour-bearing rats during AC7700 treatment. (A–C), mean tumour area±s.d. (mm²) in the maximum sections of the left lung, the left lobe of the liver, and the myocardium of the left ventricle. (D) and (E) mean tumour weight±s.d. (g) in the subcutis and lymph node metastases. Tumour size in the lung and subcutis in the AC7700-treated group was significantly smaller than that in the 0.9% NaCl-treated group (lung tumour, P=0.0228; subcutaneous tumour, P<0.0001). The number of lymph node metastases was significantly smaller in the AC7700-treated group compared with the control group (P=0.0054). There was no significant difference in body weight between the two groups.

Figure 5

Blood flow stasis in tumour capillaries caused by 10 mg kg⁻¹ AC7700. (A) before AC7700 administration; (B) 3 min later; (C) 5 min later; (D) 20 min later. Tumour blood flow in this region completely stopped approximately 2 min after the AC7700 administration. Original magnification, ×100. Bar, 100 μm.

Figure 6

Contraction of a host arteriole and a tumour-feeding arteriole caused by 10 mg kg⁻¹ AC7700. (A) before AC7700 administration; (B) 5 min later; (C) 30 min later; (D) 3 h later. Tumour blood flow completely stopped 30 min after i.v. administration of AC7700. Note that an arteriole (white arrow) underwent marked contraction in response to AC7700, and a feeding arteriole (asterisk) into a tumour disappeared from view. Original magnification, ×20. Bar, 250 μm.

Figure 7

Inhibition of SLC tumour growth caused by AC7700 in the tumour developing in a transparent chamber. Tumour size at the start of observation was defined as 100%. During the 48 h after a single i.v. administration of 10 mg kg⁻¹ AC7700, tumour size did not change at all (solid circle) (n=4). In contrast, tumours in the control group continued to grow (open circle) (n=4). Tumour area doubling time was 41.7±11.4 h.

Figure 8

Typical finding of growth inhibition caused by AC7700 in an SLC microtumour developing in a transparent chamber. (A) before administration of 10 mg kg⁻¹ AC7700 administration; (B) 3.5 h after administration of AC7700; (C) 25 h later; (D) 48 h later; (E) histology 48 h later. Tumour blood flow completely stopped at 1 h after a single i.v. administration of AC7700. The whole region of the tumour, with a diameter of 2.5 mm, became necrotic. Tumours stopped growing completely during the 48-h observation period. Histological study (E and F, H&E stained) certified the tumour (shown on the right side) as necrotic. Original magnification: A–D, ×20; E, ×200; F, ×400. Bars: A–D, 500 μm; E, 100 μm; F, 50 μm.

Figure 9

Typical finding of growth of an SLC tumour developing in a transparent chamber in the control group. (A) 0 h; (B) 24 h later; (C) 48 h later. The tumour never stopped growing during the observation period. Original magnification, ×20. Bar, 500 μm.