Double-stranded DNA breakage signaling cell death through the p53 pathway is probably the best-described pathway for radiation-induced apoptosis. BAX may also be induced by radiation via the death receptor signaling pathway and subsequent ceramide induced protein kinase activation (30
). Pharmacological inhibition of Bcl-2 has shown efficacy in a variety of co-therapeutic regimens for cancer using both in vitro
and in vivo
tumor models (13
). The effect of Bcl-2 inhibition and radiation on the endothelial fraction of tumors is less well understood. Endothelial apoptosis, senescence and proliferation are mediated by ionizing radiation under varying conditions and through clearly different mechanisms (31
). In the present study, we demonstrate the cooperative anti-tumor effects of a novel Bcl-2 inhibitor and radiotherapy on a tumor model with humanized tumor endothelium.
There is limited literature regarding the effects of ionizing radiation on endothelial cells alone and the data that has been presented is occasionally contradictory. Here, we observed little effect on proliferation when microvascular cells were exposed to ionizing radiation up to 10 Gy. It was recently demonstrated that 4-8 Gy ionizing radiation induced a senescent phenotype in proliferating bovine aortic endothelial cells and human umbilical vein endothelial cells, but not in confluent monolayers (33
). Interestingly, whilst they observed significant reduction in DNA replication, cell proliferation and invasion capabilities, they found no significant effects on their ability to form sprout-like tubes on Matrigel. Here, a significant reduction was observed in the ability of HDMEC to form sprouting structures in 3-D collagen matrices. The collagen matrix sprouting assay involves both migration and invasion over an extended period (2-8 days), unlike the Matrigel assay which is a rapid (6-24 hours) primarily migration based assay. The inclusion of an invasive element may account for the observed differences between the two studies. Indeed, a recent study showed some significant inhibition of HUVEC sprouting on a Matrigel bed (34
). The combination of TW-37 (0.5 μM) and radiation (1 Gy) significantly inhibited endothelial cell sprouting compared to either treatment alone. This suggested the potential for both treatments to cooperate via an anti-angiogenic mechanism. The combination effects of radiation and TW-37 on cell cycle are more difficult to understand. Radiation alone produces an expected G2
block and TW-37 has little noticeable effect until the concentration reaches 0.5 μM, still below a concentration for which TW-37 induces apoptosis or inhibits proliferation of endothelial cells. From 0.5 μM to 5 μM, the G1
phase fractions of cells fall while the proportion of cells in S-phase rises. It is unclear how this effect alters the fate of cells as, consistently, 5-10% of these cells will undergo apoptosis and the greater majority will cease proliferating (11
Similarly to previous studies of TW-37 and angiogenic function (11
) the concentrations of TW-37 that do not induce apoptosis still had an anti-proliferative effect that was sufficient to sensitize endothelial cells to radiation mediated growth inhibition in a clonogenic assay. This was in contrast to the SRB-based proliferation assay, which demonstrated no such sensitizing effect. Cell proliferation or cytotoxicity assays generally measure the whole population of cells in a well, plate or tube. The clonogenic assay is rarely used in endothelial cell analysis, however it serves a useful purpose here in determining individual endothelial cell survival, as shown (35
). That a sensitizing effect was observed in assay of individual cells and not in assay of the population as a whole suggested that there is a sub-population of endothelial cells that are selectively responsive to the radio-sensitizing effects of TW-37. We have recently demonstrated that 22% of endothelial cells are required to undergo apoptosis to cause a significant reduction in blood vessel density (37
). This suggests that even a small sub-population of endothelial cells sensitized to radiation may potentially have a significant effect on the tumor vasculature.
Sub-maximal doses of TW-37 and radiation were used intentionally for in vivo
studies to allow for observation of combination effects on the head and neck tumor xenografts. Radiation doses as low as 0.8 Gy administered for 5 consecutive days abolished tumor growth. Interestingly, the SCID-mouse xenograft model used in this study was consistently more sensitive to radiation in every experiment performed than might be expected for other immunodeficient murine models. The radiation exposures were carefully determined and performed by an experienced radiologist. Ultimately, 0.8 Gy administered for 3 days was used in our studies. It allowed a transient, but significant, decrease in tumor growth rate. Likewise, 60 mg/kg in combination with a chemotherapeutic regimen was necessary to cause growth inhibition on a lymphoma xenograft model (38
). Here, the tumor inhibitory effect persisted in the combination group with metronomic TW-37 over two weeks after cessation of treatment. These data supports the use of a metronomic administration of the Bcl-2 inhibitor as an effective dosing regimen that shows low toxic effects.
Pathological assessment of tumor status immediately after final treatment reflected the observed long-term tumor growth patterns. The cohesive pattern of tumor growth presented by the TW-37 and TW-37 plus ionizing radiation groups has a more favorable prognosis than the diffuse growth presented by specimens from radiation alone and vehicle control. It is widely described that tumors invading with pushing borders are less aggressive than tumors showing a non-cohesive front and diffuse spread with tiny strands or single cells (27
). Additionally, invasive fronts are typically correlated with metastases (27
). These data strengthen the argument for usage of low dose TW-37 as a positive tumor modifier.
The present study demonstrates the ability of TW-37 to sensitize endothelial cells to the effects of low dose radiation. The results further suggest that this effect may derive from a sensitive sub-population of endothelial cells that respond to TW-37 treatment and might contribute to the significant reduction of tumor blood vessel density seen in vivo. The anti-angiogenic effects of radiation demonstrated here compliment the known anti-tumor cell effects of both TW-37 and radiation in vitro and may account for an enhanced anti-tumor effect for the combination therapy in vivo. These results suggest that head and neck patients might benefit from the combination of a metronomic regimen of TW-37, or related drugs, with traditional radiotherapy.