Since 1978, local radiotherapy, administered after debulking surgery, has been a mainstay of standard treatment of GBM patients [79]. Although radiotherapy results in excellent local control and cure rates in most solid tumors [87], the efficacy of this treatment modality in GBM is extremely limited, resulting only in disease stabilization for a few months [79,80,7]. Almost all GBM patients (99%) develop fast disease progression and tumor recurrence within or imme-diately adjacent to the high-dose radiation (60 Gy) volumes [88,89], whereas local recurrences are also described after very high doses such as 90 Gy [90].
Therefore, GBM, by nature one of the most radioresistant tumors, represents a major challenge in neuro-oncology.
Detailed information about molecular mechanisms of radioresistance of GBM is not known. The basis of radioresistance may involve many tumor cell and surrounding microenvironment processes, including changes in growth factors and their receptors, different signaling and apoptotic pathways and DNA repair mechanisms [91–95]. Since there is an urgent need for new treatment strategies to improve the chance of survival for the patients of this fast-killing disease, precise knowledge about these resistance mechanisms is of great importance.
2.1. PARP-1 and DNA-PK
Radiotherapy causes a variety of DNA lesions, including single-strand brakes (SSB) and double-strand brakes (DSB) [96]. The lethal lesion is an unrepaired or misrepaired DSB produced as part of a complex lesion [97].
PARP-1 (Poly(ADP-ribose) polymerase 1) is an enzyme of PARP super-family that is responsible for most of PARP activity [98]. The most well-known role of PARP-1 is the detection of SSB [99]. After binding to radiation-induced SSB (damage detection), activated PARP recruits repair enzymes (X-ray repair cross-complementing group 1, DNA polymerase-β, DNA ligase III) that are involved in base excision repair (BER) pathway. Recruited enzymes process broken DNA ends, synthesize missing DNA and seal the gap in DNA [100, 101].
DNA-PK (DNA-dependent protein kinase) plays an important role in DNA DSB repair by nonhomologous end joining (NHEJ) pathway [102]. DNA-PK is a kinase that binds to DNA DSB, phosphorylates, and activates DNA-binding proteins (X-ray repair cross-complementing protein 4, DNA ligase IV) [103].
Due to interaction of these enzymes, double strand break ends are directly ligated [103,104]. Since DNA repair enzyme inhibitors enhance the cytotoxic effects of DNA-damaging agents (radiation, chemotherapy), their role in cancer therapy is increasingly explored [105].
2.2. CD133
According to the brain tumor cancer stem cell model, a subpopulation of cancer cells possesses the capacity of self-renewal, tumor formation and the capability to form progeny with a more restricted fate [106]. In GBM, several stem cell candidate markers have been explored, however, out of these, CD133 is the most studied [93,107].
CD133 is a transmembrane glycoprotein which is expressed in different type of progenitor cells, including hematopoietic stem cells. CD133+ GBM cells are considered stem cells because of their ability to self-renew, differentiate and to initiate tumor formation in vivo [108]. An injection of as few as 100 CD133+
cells has been shown to produce a tumor that could be serially transplanted and resembled phenotypically the patient’s original tumor [108].
Previous in vitro and in vivo studies have proposed that CD133+ tumor cells represent the cellular population that confers GBM radioresistance and could therefore be the source of tumor recurrence after radiation [109].
2.3. CD63
Radioresistance of GBM involves tumor-cell related changes, as well as changes that occur in tumor surrounding microenvironment. It has been reported that both constituents of tumor microenvironment, inflammatory and immune response markers are expressed in GBM [110,111]. Nevertheless, their exact role and impact on radiotherapy efficacy is not known.
CD63 is a lysosomal glycoprotein that is expressed on activated platelets, monocytes, macrophages, as well as on granulocytes, T-cells and B-cells [112].
Therefore, CD63 represents one of the markers of inflammation and immune response that might influence tumor micromilieu and thereby cancer cells.
2.4. ANGIOGENESIS, VEGFR-2
GBM is one of the most angiogenic tumors. Therefore, in recent years, the inhi-bition of tumor angiogenesis has been an extremely attractive and dominating experimental therapeutic strategy in neuro-oncology [113,114].
In GBM, at least five mechanisms by which tumors achieve neovascula-rization have been described: vascular co-option, angiogenesis, vasculogenesis, vascular mimicry, and glioblastoma-endothelial cell transdifferentiation [115].
Out of these, angiogenesis and vasculogenesis have been most extensively studied and described. During angiogenesis, blood vessels arise from sprouting and proliferation of endothelial cells from pre-existing vascular network, whereas in vasculogenesis, de novo blood vessels are formed through coloni-zation of circulating bone marrow-derived endothelial progenitor cells that are recruited to the tumor [115]. Both previously mentioned mechanisms of
neo-vascularization are largely regulated via vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR-2) [116].
Downstream effects of VEGFR-2 activation in the vascular endothelium include cell proliferation, migration, permeability and survival, resulting in neo-vascularization processes, such as angiogenesis and vasculogenesis [116]. Con-sequently, this receptor has been very attractive target in the development of antiangiogenic drugs (e.g. bevacizumab, sunitinib, sorafenib, vatalanib, van-detanib, recentin, cediranib) [116]. Unfortunately, a number of these antiangio-genic drugs (vandetanib, cediranib, sorafenib, sunitinib) have failed to show clinical efficacy in different phases of clinical trials both in newly diagnosed and recurrent glioblastoma [117–121]. Moreover, the most advanced antiangio-genic drug in glioblastoma – bevacizumab – did not get approval from The European Medicines Agency Committee for Medicinal Products for Human Use (CHMP) due to the lack of clinically relevant efficacy [122,123]. All these negative trials have caused a lot of frustration since the results do not coin-cidence with the initial expectations. The reasons of the lack of significant clinical efficacy of antiangiogenig drugs, however, are not fully elucidated.
It has been shown that tumor microenvironment influences GBM treatment outcome [95]. Whether inflammatory tumor microenvironment, which is one of the characteristic histological features of GBM, affects the expression of VEGFR-2 is not clear.