Previous reports have shown that interruption of VEGFR or PDGFR signaling can enhance the damaging effects of ionizing radiation
. For example, targeted therapy using cediranib, a small molecule VEGFR-inhibitor used in junction with radiotherapy, synergistically enhanced the growth delay of calu-6 lung xenografts and was associated with increased levels of apoptosis and necrosis in histological samples
. Cuneo et al.
 demonstrated the effectiveness of combining sunitinib with radiation for the treatment of human pancreatic adenocarcinomas. Their results revealed that sunitinib or radiation when used alone delayed tumor growth, however when combined, the delay was significantly enhanced. Similar findings were reported for Lewis carcinomas treated in vivo with the combination of sunitinib and radiation
. Thus with prior reports illustrating the effectiveness of the combination of sunitinib and radiation on both cell lines and xenograft tumors, derived from a variety of human cancers, we investigated whether it would radiosensitize three prostate cell lines; the hormone independent DU145 and PC3 and hormone dependent androgen receptor expressing LNCaPs. This was of interest because the radioresistance of prostate cancer cells potentially limits the outcome of radiotherapy for this disease and inhibitors directed at the mechanisms of resistance might be of benefit.
Western blot analysis (Figure
1) showed that DU145 and PC3 cells express one or more of sunitinib’s cellular targets, i.e. VEGFR2, PDGFR- and c-Kit. Based on this, we postulated that sunitinib would radiosensitize these two cell lines but perhaps not radiosensitize the LNCaP cell line, found to express none of the given targets. This indeed turned out to be the case when sunitinib radiosensitization was assessed by clonogenic assay (Figure
3); DU145 and PC3 cells were modestly radiosensitized and LnCaP cells were not. However, in spite of the modest radiosensitization seen using sunitinib on DU145 and PC3 cells, the reduction in SF2 values observed would be predicted to have clinical impact in a fractioned treatment protocol in prostate cancer patients.
In spite of growing interest in combining novel tyrosine kinase inhibitors (TKIs) with conventional techniques such as radiotherapy, the molecular mechanisms by which TKIs elicit their sensitizing effects remain to be elucidated
. However, generally, it appears that many if not most TKIs inhibit signaling downstream of growth factor receptors mediated by the PI3K-AKT and Ras-Raf-MEK-ERK pathways
[15, 16]. Activation of both the ERK and AKT pathways are a frequent event in prostate cancers and a strong association between the expression of these kinases and poor prognosis is often observed
[17, 18]. Thus, we tested whether sunitinib suppressed p-AKT and/or p-ERK, 2 appropriate downstream elements of the signaling pathways under investigation. The results showed that sunitinib suppressed p-ERK in un-irradiated and irradiated DU145 and PC3 cells suggesting that radioresistance in these cells lines is mediated through the Ras-Raf-MEK-ERK pathway. This is consistent with numerous reports in the literature illustrating the importance of this pathway in governing radiation response in tumor cells
Perhaps the most important mechanism for dictating the cytotoxicity of ionizing radiation involves the repair of radiation-induced DNA double strand breaks (DSBs)
. Repair of these lesions critically determines the degree of cell killing by radiation. Induction and repair of radiation-induced DSBs is commonly followed using the detection of γH2AX foci
. This assay is very sensitive and we have used it previously to demonstrate that the radiosensitizing action of other molecularly-targeted agents involves an inhibition of DSB repair detected on the basis of a prolongation of γH2AX foci in the agent plus radiation samples compared to radiation alone controls
. In the present study, however, we were unable to detect any prolongation of γH2AX foci by sunitinib (Figure
5) suggesting that sunitinib does not interfere with the repair of radiation-induced DSBs. This may not be too surprising since the degree of radiosensitization produced by sunitinib here is small compared to what was observed in our previous studies using other molecularly targeted agents. Thus, it is conceivable that sunitinib suppresses DSB repair to a small degree that is undetectable by this assay or that sunitinib radiosensitizes by some other mechanism.
Based on the experiments conducted in vitro, we hypothesized that daily sunitinib treatments concurrent with daily fractionated radiation would enhance tumor growth delay compared to radiation alone. However, sunitinib given concurrently with radiation did not prolong tumor growth delay. Conversely, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced. We conclude that, at least in this treatment protocol and tumor model, sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro or that the modest degree of radiosensitization seen in vitro cannot be observed in the in vivo model. Alternatively, the anti-angiogenic activity of sunitinib may increase tumor hypoxia when administered prior to radiation thereby decreasing radiosensitivity and offsetting any radiosensitizing effect of the drug
. This possibility is supported by previous reports showing that sunitinib and other anti-angiogenic agents may enhance tumor blood vessel distruction during fractionated irradiation
The fact that tumor growth delay was enhanced when sunitinib was given after radiotherapy was completed suggests that sunitinib may be acting on the irradiated tumor stroma and suppressing its ability to sustain regrowth of the irradiated tumor. This latter effect is consistent with previous reports illustrating enhanced tumor control when anti-angiogenic agents are applied after the completion of radiotherapy
. For example, Zips et al. reported that the adjuvant application of PTK787/ZK222584 preferentially retarded tumor growth when combined with fractionated irradiation
[24–26]. Similar findings have been reported for other anti-angiogenic agents including bevacizumab, ZD6474 and sunitinib
[11, 23, 27].
Our results demonstrate the effectiveness of sunitinib when combined with radiation for enhancing the radiosensitivity of androgen independent prostate cancer cells when treated in vitro. Although a mechanism mediating this response was not isolated, further studies into signaling functions downstream of sunitinib’s targeted growth factor receptors may ultimately provide greater insights. In the in vivo study, enhancement of tumor growth delay was not observed when sunitinib was given concurrently with fractionated radiation. However, tunor growth delay was enhanced when sunitinib treatment was initiated after the completion of fractionated radiation suggesting that sunitinib suppresses the ability of the tumor stroma to sustain regrowth of the irradiated tumor. Castrate-resistant clones can be a dilemma for radiation since the best outcomes depend on combination therapy with androgen deprivation to decrease tumor bulk locally and prevent or delay metastasis. The data submitted here and other reports in the literature suggest that the combination of TKIs such as sunitinib with radiation offers a promising approach. However, the effectiveness of such combinations may critically depend on appropriate scheduling of the agents.