Purpose

To investigate the effect of a metronomic (low dose, high frequency) small molecule inhibitor of Bcl-2 (TW-37) in combination with radiotherapy on microvascular endothelial cells in vitro and in tumor angiogenesis in vivo.

Methods and materials

Primary human dermal microvascular endothelial cells (HDMEC) were exposed to ionizing radiation and/or TW-37, and colony formation as well as capillary sprouting in 3-D collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by co-transplantation of human squamous cell carcinoma cells (OSCC3) and HDMEC seeded in highly porous biodegradable scaffolds into the subcutaneous space of immunodeficient mice. Mice were treated with metronomic TW-37 and/or radiation, and tumor growth was evaluated.

Results

Low dose TW-37 sensitized primary endothelial cells to radiation-induced inhibition of colony formation. Low dose TW-37 or radiation partially inhibited endothelial cell sprout formation, while in combination these therapies abrogated new sprouting. Combination of metronomic TW-37 and low dose radiation inhibited tumor growth and resulted in significant increase in time to failure as compared to controls, whereas single agents did not. Notably, histopathological analysis revealed that tumors treated with TW-37 (with or without radiation) are more differentiated and showed more cohesive invasive fronts, which is consistent with less aggressive phenotype.

Conclusions

These results demonstrate that metronomic TW-37 potentiates the anti-tumor effects of radiotherapy, and suggest that patients with head and neck cancer might benefit from the combination of small molecule inhibitor of Bcl-2 and radiation therapy.

Members of the Bcl-2 family play a major role in the pathobiology of head and neck cancer. We have shown that Bcl-2 orchestrates a crosstalk between tumor cells and endothelial cells that have a direct impact on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly upregulated in the tumor associated endothelial cells as compared to the endothelial cells of normal oral mucosa in patients with HNSCC. Here, we evaluated the effect of TW-37, a small molecule inhibitor of Bcl-2, on the cell cycle and survival of endothelial cells and HNSCC and on the progression of xenografted tumors. TW-37 has an IC50 of 1.1 µM for primary human endothelial cells and averaged 0.3 µM for head and neck cancer cells (OSCC3, UM-SCC-1, UM-SCC-74A). Combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, as compared with single drug treatment . Notably, while cisplatin led to an expected G2/M cell cycle arrest, TW-37 mediated an “S” phase cell cycle arrest in endothelial cells and in HNSCC. In vivo, TW-37 inhibited tumor angiogenesis and induced tumor apoptosis without significant systemic toxicities. Combination of TW-37 and cisplatin enhanced the time to tumor failure (i.e. 4-fold increase in tumor volume), as compared to either drug given separately. Collectively, these data reveal that therapeutic inhibition of Bcl-2 function with TW-37 is sufficient to arrest endothelial cells and HNSCC in the “S” phase of cell cycle, and to inhibit head and neck tumor angiogenesis.