Mechanistic investigation of Myo-Inositoltrispyrophosphate as a hypoxia modulator in combination with radiotherapy

Abstract

Cancer is a leading cause of death worldwide. Despite several treatment options such as radiotherapy, surgery, chemotherapy and molecularly targeted therapy, and combinations of those, the fight against cancer is not satisfying yet. There is no “miraculous cure” and there will never be, since “cancer” is only a broad designation of a malady that covers a huge amount of different forms and behaviors. The only thing in common is the degenerated control of cells that start to grow uncontrollable, achieved through many different mechanisms. Given that, the work on the improvement of existing methods to counteract cancer, and on finding and developing new strategies, is of great importance. Radiotherapy is one of the most commonly used treatment modalities, alone or in combination with another. It induces a complex network of secreted factors that can stimulate tumor outgrowth, dissemination, incomplete tumor regression and immune reactions. Irradiation can contribute to tumor eradication by damaging cells, respectively their DNA, in an amount that cannot be repaired anymore and will lead to mitotic catastrophe that either ends in senescence or death of the cell. DNA damage by irradiation is achieved through the formation of reactive oxygen species (ROS), generated through radiolysis. ROS are highly reactive and will lead to damage of the DNA and other macromolecules. The response and treatment outcome of irradiation is dependent on several factors, described as the 5 Rs of radiotherapy. These include the repair capacity of the cells and their intrinsic radiosensitivity, depending on their position within the cell cycle. After irradiation, the cells will redistribute within the cell cycle and repopulate in order to fill up a void created by irradiation-induced cell death. The fifth R addresses the reoxygenation between two dose fractions. Reoxygenation is crucial because oxygen plays a key role in radiotherapy. It is necessary for the stabilization (“fixation”) of damage induced by ROS. Without sufficient oxygen, the survival fraction of irradiated cells is 2.5 to 3 times higher as compared to normal oxygenated cells. For this reason, the oxygen status of the tumor is a major predictor and influencer of radiotherapy treatment outcome. There are several strategies available to increase the amount of oxygen in the blood, and therefore its delivery to a tumor, but they are still not fully gratifying. In this Master’s Thesis, a novel compound, Myo-Inositoltrispyrophosphate (ITPP), was investigated. It was developed as an effector of hemoglobin to lower the affinity of hemoglobin to oxygen. Thereby an enhanced release of oxygen e.g. in hypoxic tumors can be achieved. So far, the mode of action remains unclear. Further, ITPP has structural similarities to PIP2 and PIP3, which are second messengers upstream of the Phosphoinositide 3-kinase / Protein kinase B (PI3K/AKT) pathway, which can influence tumor growth and progression. This Master’s Thesis demonstrates that ITPP can modulate the availability of oxygen within a tumor and reoxygenate hypoxic tissue. Through the increase of oxygen, the radiosensitivity increases, leading to a significant growth delay in vivo, when radiotherapy is combined with ITPP. This was demonstrated in an efficacy-oriented experiment of A549 xenografts and in FaDu xenografts. As part of a broad mechanistic investigation, histological analysis of tumor oxygenation and of DNA double strand breaks (DSB) by γH2AX staining was performed. A significant increase in DNA DSBs in initially hypoxic tumor zones could be achieved with ITPP in combination with radiotherapy. Through irreparable damage like DNA DSBs, cells will experience radiation-induced loss of clonogenicity, primarily via mitotic catastrophe or other modes of cell death. These results suggest that a tumor growth delay achieved through a combined treatment modality of ITPP and irradiation underlies the induction and “fixation” of DNA DSBs, not only in normoxic but also in reoxygenated tumor zones due to ITPPs oxygen-modulatory effect. Furthermore, ITPP has also an impact on the PI3K/AKT pathway in vitro, as it upregulates PTEN levels and decreases pAKT concomitantly. In summary, this Master’s Thesis presents mechanistic investigations on the treatment modality of ITPP in combination with radiotherapy in vivo through the oxygen-modulating capabilities of ITPP: more ROS can be produced through irradiation, leading to the oxygen-enhancing effect within hypoxic areas of the tumor and more extended induction of DNA-double strand breaks. Therefore, ITPP is claimed as a potent radiosensitizer and neoadjuvant treatment option for hypoxic tumors.