Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers

Abstract

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G 2/M and cells with lower G(0)/G(1) DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14-27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.

Figure 1. RT-PCR (A) and western blot (B) analysis of GRP-R, NMB-R and BSR-3 in HT-29 (lane 1), HCT-116 (lane 2) and HCT-15 (lane 3) human colon cancer cell lines. cDNA products (A) and proteins (B) were of the expected size of 79 bp (43 kd), 83 bp (43 kd) and 85 bp (44 kd) corresponding to GRP-R, NMB-R and BSR-3. DNA molecular marker is presented in lane M. Negative controls in reverse transcription reaction with no RNA in reverse transcription reaction and no cDNA in RT-PCR is shown in lane 4.

Figure 2. Shows cell cycle distribution analysis of control and treated cells by flow cytometry. In control cells and those exposed to RC-3940-II (1 μM) or GRP (100 nM), 21–26% of the cells had S-Phase DNA content. In contrast cells exposed to RC-3940-II (10 μM), GRP (100 nM) + RC-3940-II (1 μM) or GRP (100 nM) + RC-3940-II (10 μM) had a significant increase in the number of cells blocked in S-phase (28–35%). There was also an increase in the number of cells with less than G₀/G₁ DNA content. These histograms indicate both an accumulation of S-phase cells and the presence of apoptotic and necrotic cells. This figure is representative of two independent experiments.

Figure 3. Effect of BN/GRP antagonist 3940-II given s.c. at a dose of 10 μg/d, 5-FU given i.p. at a concentration of 30 mg/kg BW at day 1, 8, 15 and 21, or the combination of both on the tumor volume of HT-29 (A), HCT-116 (B) and HCT-15 (C) human colon cancer cells xenografted s.c. into nude mice. Vertical bars indicate SE *p < 0.05 vs. control.

Figure 4. Effect of BN/GRP antagonist 3940-II given s.c. at a dose of 10 μg/d, irinotecan given i.p. at a concentration of 50 mg/kg BW at day 1, 8, 15 and 21, or the combination of both on the tumor volume of HT-29 (A), HCT-116 (B) and HCT-15 (C) human colon cancer cells xenografted s.c. into nude mice. Vertical bars indicate SE *p < 0.05 vs. control.