F10 Inhibits Growth of PC3 Xenografts and Enhances the Effects of Radiation Therapy

Abstract

Chemotherapy remains of limited use for the treatment of prostate cancer with only one drug, docetaxel, demonstrating a modest survival advantage for treatment of late-stage disease. Data from the NCI 60 cell line screen indicated that the castration-resistant prostate cancer cell lines PC3 and DU145 were more sensitive than average to the novel polymeric fluoropyrimidine (FP), F10, despite displaying less than average sensitivity to the widely-used FP, 5FU. Here, we show that F10 treatment of PC3 xenografts results in a significant survival advantage (treatment to control ratio (T/C) days = 18; p < 0.001; n = 16) relative to control mice treated with saline. F10 (40 mg/kg/dose) was administered via jugular vein catheterization 3-times per week for five weeks. This aggressive dosing regimen was completed with no drug-induced weight loss and with no evidence of toxicity. F10 was also shown to sensitize PC3 cells to radiation and F10 was also shown to be a potent radiosensitizer of PC3 xenografts in vivo with F10 in combination with radiation resulting in significantly greater regression of PC3 xenografts than radiation alone. The results indicate that F10 in this pre-clinical setting is an effective chemotherapeutic agent and possesses significant radiosensitizing properties.

Keywords: Fluoropyrimidine; Prostate cancer; Radiosensitization; Thymidylate synthase.

Figures

Figure 1

F10 strongly inhibits the clonogenicity of PC3 cells. (A) Structure of F10. (B) Clonogenic survival assay evaluating clonogenic survival of PC3 cells following 72 h treatment with either F10 or 5-FU at the indicated concentration (nM). No surviving colonies were observed following treatment with F10 at 1 μM (1,000 nM). In contrast, treatment with 5FU at 10 μM (10,000 nM) decreased the percent of colonies surviving relative to control by less than 50%. (C) TS activity in PC3 cells at the indicated times following treatment with F10 (10-8 M) or 5-FU (10-6 M) (* p < 0.05 vs control based on Student's two sided t-test).

Figure 2

Results of a clonogenic assay evaluating the survival fraction of PC3 cells exposed to radiation or radiation in combination with F10. (A) Graph of surviving fraction as a percent of non-treated cells. (B) Same data as in (A) but normalized for F10-only effects to determine if F10 were radiosensitizing. (* p

Figure 3

Treatment with F10 significantly reduces the growth of established PC3 cell xenografts. (A) Tumor growth curves for the four tumor groups in the study (left and right flank tumors from each treatment group were analyzed separately). Error bars indicate +/-SEM, n = 8. (B) A typical mouse in the study. Tumors were initiated by s.c. injection of PC3 cells bilaterally. Initial tumor volumes were approximately 500 mm3 in all treatment groups. The left flank tumor in each mouse was selectively irradiated using the 300 kV orthovoltage x-ray irradiator. (C) Treatment schedule for the in vivo experiment. Mice were assigned to one of two treatment groups – either F10 at 40 mg/kg dissolved in 100 μL of sterile saline or saline-only. Treatment was administered through a catheter inserted into the jugular vein. Drug was administered 3× per week for five weeks on the days indicated. A dose of 3 Gy radiation was administered to the left flank of all animals 2× per week for five weeks on the indicated days. (D) Kaplan-Meier survival curves for mice treated with F10 or with saline control. Mice were removed from the study based on tumor size in the non-irradiated flank.

Figure 4

F10 treatment does not result in weight loss greater than control treatment or cause damage to the colonic epithelium. Also, the combination of F10 + radiation results in significantly smaller final tumor size relative to treatment with radiation alone. (A) Average weights for F10-treated and control mice. Error bars indicate +/-SEM, n = 8. (B) Graph of the final mean tumor volumes for left-flank tumors in the study. Tumors treated with F10 + irradiation were significantly smaller than tumors treated with radiation. Graph depicts the mean +/- SEM (n = 8) for left flank tumors from the F10-treated and saline groups. (C) H&E section (10×) of colonic epithelium from an F10-treated mouse. (D) Colonic epithelium from a saline-treated mouse. Both mice were sacrificed as a result of tumor burden to the non-irradiated flank. There is no deterioration of the colonic epithelium in mice from treated with F10.

Figure 5

H&E sections (4×) from a left-flank (irradiated) tumor from an (A) F10-treated animal; and (B) Saline-treated animal. Irradiated tumors from the F10-treated group displayed marked hypocellularity relative to saline-treated animals. (C) H&E section from the right-flank (non-irradiated) tumor from an F10-treated animal and (D) from the right-flank of a saline-treated animal. While animals from both groups were sacrificed based on volumes in the right-flank tumors, tumors from the saline group displayed marked necrosis. Animals from the F10-treated group were sacrificed, on average, 18 days later than animals in the saline group (see Figure 3).