Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models

Abstract

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH-302) is a prodrug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross-linking effector, Br-IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA-MB-231-TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer-induced bone destruction was measured using micro-CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

Keywords: Breast cancer; TH-302; chemotherapy; evofosfamide; hypoxia; tumor growth.

Figure 1

Activity of evofosfamide against breast cancer, epithelial cells, and fibroblasts in vitro. (A) Six breast cancer cell lines were seeded in 96‐well plates at 1 × 10⁴ cells per well and treated with increasing doses of evofosfamide in normoxic (21% O₂) and hypoxic (1% O₂) conditions for 48 h. (B) The breast cancer cell line MDA‐MB‐231‐TXSA was treated with an increasing dose of evofosfamide under normoxic (21% O₂) and in various hypoxic conditions from 0% to 5% O₂ for 24 h. (C) Evofosfamide reduced cell viability in both epithelial breast cell lines MCF‐10A and MCF‐12A selectively under hypoxic conditions (1% O₂) 48 h after treatment. Dermal and mammary fibroblasts were relatively resistant to evofosfamide under the same conditions. Cell viability of all cell lines was assessed by crystal violet staining. (D) DAPI nuclear fluorescence stain of untreated MDA‐MB‐231‐TXSA cells showing nuclei homogenously stained. Cells treated with 50 μmol/L evofosfamide under hypoxic conditions for 24 h exhibit changes in the nuclei consistent with the induction of apoptosis. Data points show means of quadruplicate results from a representative experiment, repeated at least twice and presented as the mean ± SD of quadruplicate wells and expressed as a percentage of the number of control cells.

Figure 2

Evofosfamide‐induced apoptosis in breast cancer cells in vitro. (A) Four breast cancer cell lines were seeded in 96‐well plates at 1 × 10⁴ cells per well and treated with evofosfamideat 50 μmol/L or coincubated with the broad specificity caspase inhibitor z‐VAD‐fmk (50 μmol/L). To exclude possible toxic effects of the inhibitor, cells were also treated with the inhibitor alone under normoxic and hypoxic (1% O₂) conditions. Cell lysates were used to determine caspase‐3‐like activity, using the caspase‐3‐specific fluorogenic substrate, zDEVD‐AFC, and cell viability was assessed via crystal violet staining. Data points show means of quadruplicate results from a representative experiment, repeated at least twice; bars ± SD.

Figure 3

Apoptotic signaling of evofosfamide as a single agent against MDA‐MB‐231‐TXSA cells. (A) MDA‐MB‐231‐TXSA‐TGL cells were seeded at 2 × 10⁶ per T25 flask and were treated with evofosfamide at 50 μmol/L under normoxic (21% O₂) and hypoxic (1% O₂) conditions. Cells were then lysed and protein lysates were collected at 0, 6, 12, 24, and 48 h after treatment. Cell lysates were analyzed by polyacrylamide gel electrophoresis and transferred to PVDF membranes for immunodetection as described in Materials and Methods section. Evofosfamide treatment resulted in cleavage and prominent activation of the initiator caspase‐8 followed by caspase‐9, and caspase‐3 and the concomitant processing of BID and PARP protein, only at the later time point of 48 h, indicating that these changes were an effect rather than a cause of evofosfamide‐induced apoptosis. cIAP1/2 levels were significantly reduced with evofosfamide treatment, under hypoxic conditions at the 24‐ and 48‐h time points, whereas the levels of XIAP, BAX, and Bcl‐2 remained unchanged. (B) A robust reduction in PI3 kinase and phosphorylated AKT under hypoxic conditions after treatment with TH‐302 at 24 and 48 h. However, no significant changes in the levels of either phosphorylated or total MAPK were detected following treatment.

Figure 4

Paclitaxel cooperates with evofosfamide to reduce MDA‐MB‐231‐TXSA‐TGL orthotopic tumors in vivo. Mice were treated as described in Materials and Methods section and imaged weekly using the Xenogen IVIS 100 bioluminescence imaging system. (A) Representative whole body bioluminescent images of the mammary tumors of a single animal from each group and (B) the line graph, showing average tumor signal over time, expressed as mean photon counts per second during the course of the experiments are shown. Animals receiving treatment with evofosfamide and paclitaxel as single agents showed a significant delay in tumor growth. In addition, all mice receiving the combination of evofosfamide and paclitaxel showed a further delay of tumor growth when compared with each agent individually. Data shown in each case are the average bioluminescent imaging from all animals in that group: points are means ± SEM.

Figure 5

Paclitaxel cooperates with evofosfamide for increased anticancer efficacy against the breast cancer line MDA‐MB‐231‐TXSA‐TGL in vivo. MDA‐MB‐231‐TXSA‐TGL cells were injected directly into the tibial marrow cavity of 4 week female athymic mice, allowed to establish for 7 days, as described in Materials and Methods section, mice were imaged weekly using the Xenogen IVIS 100 bioluminescence imaging system. (A) Representative whole‐body bioluminescent images of a single mouse from each group during the course of the experiment are shown. (B) The line graph represents average tumor signal over time, measured as photon counts per second. Evofosfamide and paclitaxel as single agents reduced tumor growth, and in addition, the combination of both resulted in an even greater inhibition of tumor growth. (C) Quantitative assessment of total bone loss (%) comparing the tumor‐bearing tibiae of each group to the contralateral tibiae and the qualitative 3‐D micro CT images show the osteolytic nature of the MDA‐MB‐231‐TXSA‐TGL cell line, which was reduced by paclitaxel and the combination of evofosfamide and paclitaxel. (D) Representative H&E‐stained tibial sections from mice showing an inhibition of intra‐ and extramedullary growth with evofosfamide, paclitaxel, and the combination of both.