Several pharmacological agents such as hypoxic cell sensitizers and pyrimidine analogues have been investigated in attempts to increase the tumoricidal effects of radiotherapy (3
). However, these approaches have shown little success because efficacious levels could not be achieved in vivo
without undue toxicity. Furthermore, the lack of methods to measure tumor hypoxia directly during these treatments has limited the complete understanding of their effect and optimization. Cell cycle arrest also undermines the therapeutic outcome of modalities such as chemotherapy and radiotherapy. Accordingly, cell cycle checkpoint inhibitors are being investigated in the hope that they will enhance cell killing after DNA damage by preventing cell cycle arrest, thereby driving cells to mitotic catastrophe. An added advantage with inhibitors such as UCN-01 is that extremely low concentrations (10–100 nM
) could be used to enhance therapeutic outcome (14
). Surprisingly, in spite of its inhibitory effect on various malignant cells, there have not been attempts to investigate its potential application in a multimodal approach along with external-beam radiotherapy.
The RIF-1 tumors were hypoxic on day 0, and no change in tumor pO2
was observed in the control and UCN-01 groups. Both UCN-01 and radiation treatments delayed tumor growth, but the growth delay was greater in the group treated with 20 Gy. However, neither treatment decreased tumor volumes. The baseline tumor pO2
and the increase on days 3 to 5 after irradiation are in agreement with our earlier findings (10
). Several different mechanisms have been suggested for reoxygenation after irradiation, such as reduced oxygen consumption, migration of hypoxic cells to an oxygenated state, and improved microcirculation (27
A significant inhibition of tumor growth was observed in tumors treated with UCN-01/20 Gy accompanied by a significant increase in tumor pO2. The tumors treated with 20 Gy/UCN-01 also had significant growth inhibition and an increase in tumor pO2. Tumor oxygenation observed with these treatments is likely due to increased tumor cell killing, reduced oxygen consumption, and a decrease in interstitial pressure with tumor shrinkage.
The UCN-01/20 Gy and 20 Gy/UCN-01 groups showed an increase in tumor pO2 with decreasing tumor volume ( and ). Therefore, changes in tumor pO2 could be used as a potential marker for tumor inhibition. To our knowledge, this is the first report of the changes in tumor pO2 in a multimodal approach of UCN-01 with radiotherapy. The noteworthy increase in tumor pO2 from a pretreatment hypoxic level to a well-oxygenated level is likely to have a significant impact in fractionated radiotherapy protocols.
UCN-01 alone affected tumor growth and inhibition irrespective of the sequence of UCN-01 and radiation. This suggests that the effect may not be due to targeting Chk1. UCN-01 is also a non-specific kinase inhibitor that, in addition to Chk1, inhibits other kinases including PKC, PDK1 and C-TAK1 (30
). UCN-01 was originally developed as a PKC inhibitor. When it was tested in animal models as a single agent, it induced tumor growth delay (32
). In its combined application with DNA-damaging modalities, the sequence of treatment is important and is related to the p53 status of the tumor. Irradiation of p53 wild-type tumors such as RIF-1 is expected to induce a p53 response that protects the tumor from subsequent Chk1 inhibition. However, if Chk1 is inhibited during the time of DNA damage, the cell killing is enhanced because the p53 response has not been induced (35
). Irrespective of the underlying mechanism, the tumor inhibition is significant and could be used to induce tumor regression in several malignancies.
In summary, these results provide evidence that UCN-01 in combination with radiotherapy could provide an effective tool for tumor inhibition. Repeated tumor pO2 measurements using EPR oximetry provide crucial data on tumor pO2 during this multimodal approach. The observed changes in tumor pO2 could be used as a marker to predict outcome. The changes in tumor pO2 also could be used to schedule irradiations at times of increased tumor oxygenation to optimize the outcome of a fractionated regimen. Furthermore, tumor pO2 could be used to identify responders and non-responders at early times during the treatment, which will allow clinicians to prescribe alternate therapies for non-responders.