Dipalmitoylation of radicicol results in improved efficacy against tumor growth and angiogenesis in vivo

Abstract

Tumor-related angiogenesis is likely to be a potential target for the treatment of cancer. One key to develop this angiostatic strategy would be to find useful angiogenesis inhibitors. Here we report the effects of radicicol, a microbial angiogenesis inhibitor that we previously identified using the chorioallantoic membrane assay, and its novel analog, 14,16-dipalmitoyl-radicicol, on tumor angiogenesis and growth. As expected for agents containing a penolic hydroxyl group, systemic administration of radicicol had little or no effect on neovascularization triggered by a M5076 mouse tumor cell line or a RMT-1 rat mammary carcinoma cell line established from autochthonous rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene in a mouse dorsal air sac assay system. The agent did not show growth-inhibitory activity against either transplantable M5076 tumors or autochthonous 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. In contrast, 14,16-dipalmitoyl-radicicol potently suppressed tumor angiogenesis and growth in these experimental models. Furthermore, the analog significantly prolonged the survival rate of M5076-implanted mice. Although not stronger than radicicol, it dose-dependently inhibited embryonic angiogenesis in the chorioallantoic membrane assay, the dose required for half-maximal inhibition (ID(50)) value being 23 microg (27 nmol) per egg, and showed concentration-dependent antiproliferative activity against microvascular endothelial cells in vitro. These data suggest that 14,16-dipalmitoyl-radicicol is a promising antitumor agent with antiangiogenic activity.

Figure 1

(a) Chemical structures of radicicol (RAD) and 14,16‐dipalmitoyl‐radicicol (DP‐RAD). (b) Antitumor activity of RAD and DP‐RAD against the early stage of transplantable M5076 tumors. Control, RAD and DP‐RAD were administered intraperitoneally four times every 4 days from day 6 after tumor cell inoculation. Points are mean ± SD for seven mice. *P < 0.05; **P < 0.001 compared to the vehicle‐treated control group.

Figure 2

(a) Antitumor activity of dipalmitoyl‐radicicol (DP‐RAD) against the advanced stage of transplantable M5076 tumors. Mice bearing the advanced stage of M5076 tumors received control, one cycle or two cycles of treatment with DP‐RAD (200 mg/kg, three times every four days, intraperitoneally). Points are mean ± SD for five ot seven mice. **P < 0.001 compared to the vehicle‐treated control group. ^# P < 0.05, ^## P < 0.001 compared to one‐cycle treatment group. (b) Survival of mice bearing the advanced stage of transplantable M5076 tumors after one or two cycles of treatment with DP‐RAD (200 mg/kg, three times every 4 days, intraperitoneally).

Figure 3

Antitumor activity of radicicol (RAD) and dipalmitoyl‐radicicol (DP‐RAD) against autochthonous rat mammary tumors. Rats bearing mammary carcinomas of approximately 10 mm in diameter received intraperitoneal injection of RAD or DP‐RAD six times every 4 days. Tumor volume was expressed as a percentage of the starting volume (100%). Points are mean ± SD for 14–25 tumors in six or seven rats. *P < 0.05 compared to the vehicle‐treated control group.

Figure 4

(a) Effect of radicicol and dipalmitoyl‐radicicol on M5076 cell‐induced angiogenesis in the mouse dorsal air sac assay system. (A) Mice with an implanted chamber containing phosphate‐buffered saline (PBS) were treated with the vehicle (i.e. negative control group); mice with an implanted chamber containing M5076 cells (2.5 × 10⁷) were administered the vehicle (B) (i.e. positive control), radicicol (100 mg/kg) and dipalmitoyl‐radicicol (200 mg/kg) on days 0 and 4 after the implantation of chambers. Four mice per group were killed on day 5 to assess the antiangiogenic effects. (b) Inhibition of M5076 cell‐induced angiogenesis by dipalmitoryl‐radicicol, but not radicicol. Groups A and B received chambers containing PBS and M5076 cells, respectively, and each group was treated with the vehicle. Groups C and D received M5076 cell‐containing chambers, and were treated with radicicol (100 mg/kg) and dipalmitoyl‐radicicol (200 mg/kg), respectively, on days 0 and 4 after the implantation of chambers. Four mice per group were killed on day 5 to assess the antiangiogenic effects. *P < 0.05 between the indicated groups.

Figure 5

(a) Effect of radicicol and dipalmitoyl‐radicicol on 5‐day‐old chorioallantoic membrane (CAM) angiogenesis. The CAM angiogenesis assay was carried out with an empty methylcellulose pellet alone (i.e. control) (A), a methylcellulose pellet containing radicicol (0.5 µg per egg) (B) or dipalmitoyled radicicol (100 µg per egg) (C). The results shown are for a representative experiment. Arrowheads indicate an avascular zone. (b) Dose‐dependent inhibition of 5‐day‐old CAM angiogenesis by dipalmitoyl‐radicicol. The points indicate the frequencies (%) of avascular zones showing an antiangiogenic response. The values in parentheses are the numbers of CAM examined. *P < 0.05; **P < 0.001 compared to the value for control CAM (n = 25) not showing an avascular zone.

Figure 6

Antiproliferative activity of radicicol (RAD) and dipalmitoyl‐radicicol (DP‐RAD) against microvascular endothelial cells and RMT‐1 rat mammary tumor cells in vitro. The proliferation of human dermal microvascular endothelial cells (HDMEC) or RMT‐1 cells was determined in the presence of various concentration of RAD or DP‐RAD. Values are expressed as a percentage of control vehicle alone. Points are mean ± SD (n = 3 for HDMEC; n = 4 for RMT‐1 cells).