Browsing by Subject "Chemotherapie"

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Hyperthermie steigert die Zytotoxizität von Cisplatin und Doxorubicin durch Hemmung der Poly(ADP-Ribosyl)ierung und der damit einhergehenden Verhinderung der Replikationsblockade
(2015) Schaaf, Lea; Aulitzky, Walter E.
Peritoneal carcinomatosis describes widespread metastases of cancerous tumors in the peritoneal cavity particularly arising from ovarian and colon cancers. For long time this far advanced tumor disease was considered extremely difficult to treat. Therefore, only palliative measures were carried out in most cases. This has changed significantly in recent years with the introduction of multimodal treatment options consisting of a complete macroscopic tumor reduction (CRS) followed by an intraoperative intraperitoneal hyperthermic chemotherapy (HIPEC). It is undisputed that this local chemotherapy application allows treatment with much higher drug concentrations as compared to the systemic therapy. However, the role of hyperthermia in this multimodal approach has not been fully clarified so far. There is still no clinical study available showing a clear benefit of elevated temperature in the intraperitoneal chemotherapy. Largely unknown is also which level of temperatures are really needed for an elevated cytotoxicity of chemotherapeutics. Furthermore, the molecular mechanisms behind this synergistic effect are poorly investigated. To answer these questions, an in vitro model was established mimicking the situation of the HIPEC procedure as closely as possible. This model allowed to define a very precise temperature threshold of 40°C. An effective increase in cytotoxicity of cisplatin and doxorubicin was only observed at temperatures of 40°C or above. Importantly, this temperature threshold was also of clinical relevance. Patients who reached this temperature over at least 40 minutes at two sites in the abdominal cavity, namely omental bursa and pelvis, showed a significantly increased overall and progression free survival. In-vitro hyperthermia leads to an increased intracellular concentration of doxorubicin. Interestingly, however, the synergy of hyperthermia with doxorubicin was observed even after reducing the drug concentrations to values which are also reached at 37° C. Together with the finding that hyperthermia had no effect on the amount of DNA-cisplatin adducts, these results clearly indicate that the increased intracellular drug accumulation is not the predominant mechanism behind the synergistic effects of chemotherapeutics and hyperthermia. Rather a compromised repair of cisplatin and doxorubicin induced DNA damages upon hyperthermia treatment could be identified to be important for the observed effects. This significantly delayed DNA repair depends on inhibition of the poly(ADR-ribosyl)ation (PARylation) by hyperthermia. Interestingly, hyperthermia selectively increased the efficiency of cytotoxic agents that induce PARylation. The hypothesis that inhibition of PARylation plays a key role for the synergy between hyperthermia and chemotherapy is further supported by the finding that the treatment with specific pharmacological PARP inhibitors resulted in a comparable elevation of cisplatin and doxorubicin induced cytotoxicity. In this respect PARylation could be identified as a molecular marker for a preclinical substance-screen to identify drugs acting together with hyperthermia. In addition, these in-vitro results for the first time show that there are alternatives to the hyperthermic treatment, which is associated with considerable side effects. Thus, intraperitoneal chemotherapy could be combined with systemic PARP inhibitor pre-treatment. Clinically approved specific PARP inhibitors are far better tolerated by patients than hyperthermia. Hyperthermia mediated inhibition of PARylation led to an increase in the percentage of cells with DNA double-strand breaks (DSB). Interestingly, the results of this work indicate a hyperthermia-induced switch from HR to the error-prone NHEJ. In combination with hyperthermia there was a significant increase in P-53BP1 foci formation, while both the percentage of cells with Rad51 foci and Rad51 protein level remained unchanged. The finding that these DSBs occur only in S-phase cells points to a replication-associated mechanism. In fact, in the framework of this work it could be demonstrated for the first time that drug-induced stalled replication forks are circumvented by the hyperthermia-mediated inhibition of PARylation. The unhindered progression of replication forks upon inhibition of PARylation by hyperthermia or pharmacological PARP inhibition presumably results in the increase of DNA DSBs as well as in the significantly reduced long-term-survival
Role of reactive oxygen species in anti-cancer treatment: Investigations in 2-methoxyestradiol chemotherapy and 5-aminolevulinic acid based photodynamic therapy combined with hyperthermia
(2003) Lambert, Christine; Frank, Jürgen
The thesis deals with two different ROS-generating anti-cancer treatments: chemotherapy with the endogenous estrogen metabolite 2-methoxyestradiol and 5-aminolevulinic acid based photodynamic therapy. Both treatments were investigated with the rat DS-sarcoma model, which can be used in vitro and in vivo. It the first part, it could be shown that 2-methoxyestradiol induces apoptosis in DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to the mitochondria was identified as initial apoptotic event, followed by a decrease in mitochondrial transmembrane potential and the release of AIF out of the mitochondria. In addition, upregulation of FasL and TNFalpha by 2-ME, two death receptor ligands, was observed. Although, 2-ME administration resulted in activation of caspases, pan caspase inhibitor Z-VAD-FMK could not block 2-ME induced apoptotic cell death pointing to a caspase-independent mechanism. Furthermore, an increase in formation of reactive oxygen species was observed after 2-ME treatment. However, supplementation with different antioxidants could not decrease the toxic effect of 2-ME. This finding may indicate, that reactive oxygen species are not involved in apoptosis induction, rather they are a consequence of mitochondrial damage. In vitro and in vivo combination of 2-ME with another ROS-generating treatment resulted in a synergistic anti-tumour effect. In the second part of the thesis anti-tumour effects of 5-aminolevulinic acid based photodynamic therapy combined with simultaneous hyperthermia was investigated. Analysis of apoptosis associated nuclear changes clearly demonstrated the high efficiency of this treatment regime. Formation of reactive compounds (e.g. ROS, nitrogen monoxide, peroxynitrite) which is mainly responsible for toxicity of PDT, could be assessed in the shape of massive protein nitrosylation in tumours treated with PDT alone or the combined treatment. Detection of decreased amounts of heat shock proteins (HSP70 and HO-1) which protect tumour cells against damaging influences, lowered glutathione levels and reduced MMP-activity indicate an increase in degradation of proteins. This phenomenon may be caused by excessive generation of ROS. Taken together, the presented studies could demonstrate the high benefit of combining 2-ME resp. ALA-PDT with hyperthermia (or other ROS-generating therapies), which make them interesting candidates for future clinical applications.
Untersuchungen des Einflusses von Chemotherapie auf Tumor-assoziierte Fibroblasten bei Karzinomen der Lunge und Brust in vivo und in verschiedenen ex vivo Modellen
(2009) Sonnenberg, Maike; Blum, Martin
Carcinomas are complex tissues in which the genetically altered epithelial tumor cells interact with their microenvironment, which plays a pivotal role in growth and survival of the whole tumor. Therefore, also the response of the tumor stroma may be important for the therapeutic success. The aim of the study was to clarify how tumor-associated fibroblasts (TAFs) respond to chemotherapy. We established a grading system for the activity of the TAFs based on cell counts and cell morphology within neoadjuvant treated breast cancer specimens. We compared both the stromal compartment and the tumor compartment before and after therapy to evaluate the response to chemotherapy. In this in vivo study a response of the stromal compartment was detected in samples with a high stromal activity grade before chemotherapy. To investigate the direct, more acute response of TAFs we established different ex vivo systems. On the one hand, the response to chemotherapy was tested in primary isolated TAFs from tumor tissue. On the other hand, a tissue slice culture was established to investigate the direct effect of the chemotherapy on TAFs and tumor cells within their intact microenvironment. For the cell culture experiments freshly isolated TAFs from breast and lung tumors were used. The chemosensitivity to Cisplatinum and Paclitaxel of the primary isolated TAFs was determined and compared with a panel of established human tumor cell lines. The TAFs turned out to be resistant to Paclitaxel, whereas they showed a heterogeneous response to Cisplatinum. One reason for this heterogeneous response to Cisplatinum could be the existence of somatic mutations and/or polymorphisms within DNA-damage response genes. Somatic mutations within the p53 tumor suppressor gene play a critical role in the response of tumor cells to chemotherapeutics. In this study, however, no somatic mutation was detected in any of the TAFs from lung. In other studies, different polymorphisms could be correlated with the sensitivity to cytotoxic stress. In this study, a non-significant trend towards the chemosensitivity of TAFs from lung carcinomas and the p53-Arg72Pro polymorphism could be observed, whereas the ERCC1-Asn118Asn und Mdm2-T/G309 polymorphisms had no influence on the chemosensitivity. The chemosensitivity of the TAFs was also determined within their microenvironment on the basis of tissue culture experiments performed with breast and lung carcinomas. Tumor tissue slices from breast carcinomas were treated for 72 h with Paclitaxel, whereas the tissue slices from lung carcinomas were treated with Cisplatinum. To investigate the response of the different cell types to the tested chemotherapeutic drugs within the intact microenvironment, the tissue slices were fixed and stained immunhistochemically for proliferative active and dead cells. Half of the cultivated tissue slices from breast carcinomas showed a decrease in cell counts of proliferating cells, whereas half of the cases showed an increase of dead cells after the treatment with Paclitaxel in tumor and stroma cells. The tissue slices of the lung carcinomas showed a decrease in proliferating tumor cells after Cisplatinum treatment in comparison to the untreated controls. The stroma compartment showed no proliferative activity so one could not expect a response regarding a decrease of proliferating stroma cells. Two cases showed a response to Cisplatinum regarding an increase in cell death in both cell compartments. Short term exposure to Paclitaxel led to a parallel reaction of both tumor and stromal cells in tissue culture experiments, whereas isolated TAFs from breast tumors turned out to be resistant to Paclitaxel. In contrast, isolated TAFs from lung carcinomas are significantly more sensitive to Cisplatinum than TAFs within their intact microenvironment. Consequently, the microenvironment of the tumor plays a crucial role in chemosensitivity. The central role of the microenvironment for response of TAFs to cytotoxic drugs is also demonstrated by the results obtained with lung cancer tissues. These observations indicate that, in addition to intrinsic factors, the microenvironment determines the sensitivity of TAFs to cytotoxic therapy. In summary, this work demonstrates for the first time, not only the tumor cells but also the stromal cells are an important target for the chemotherapy. Intrinsic and extrinsic factors play an important role in chemosensitivity. On the one hand, the tumor microenvironment is responsible for therapy response of TAFs and on the other hand the genotype may play a crucial role in chemosensitivity. Both, tumor and stroma cells react in parallel to chemotherapy, so you can assume that both cell types are responsible for the chemosensitivity. Some therapeutic approaches could be the inhibition of either the tumor or stromal cell compartment to increase the chemosensitivity of the whole tumor.