Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin. Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells. Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.

Testicular germ cell cancer (TGCC) has been increasing in incidence over recent decades, and is currently the most common malignancy amongst young men resulting in significant morbidity. These tumours are believed to arise from premalignant germ cell neoplasia in situ (GCNIS) cells, which originate from the aberrant germ cell differentiation from gonocyte to spermatogonia during fetal/early postnatal life. GCNIS cells remain dormant in the testis until puberty when they are activated to become tumours. Therefore, GCNIS cells remain in a pre-invasive stage during early childhood and early adulthood prior to the development of a seminoma or non-seminoma TGCC. GCNIS cells are phenotypically similar to gonocytes with expression of stem cell/early germ cell markers including OCT4, PLAP and LIN28. Furthermore, proteins which are expressed in more mature germ cells (spermatogonia) such as MAGE-A4 have also been shown to be expressed in GCNIS cells and these studies have indicated that GCNIS cells are a heterogeneous population in terms of protein expression profile. The relationship between the protein expression profile of individual GCNIS cells populations and their oncogenic potential has not been fully explored. GCNIS cells are located in the seminiferous tubules supported by somatic Sertoli cells. These cells have been previously reported to exhibit an immature protein expression profile in GCNIS tubules from patients with testis cancer, suggesting that the germ stem cell niche in GCNIS tubules resembles that of a fetal one. Associations between Sertoli cell maturation and GCNIS progression into tumour formation has not been fully investigated. Oncogenes are key players in the regulation of oncogenic potential of cancer cells. Gankyrin is an oncogene that has been shown to down-regulate OCT4, and interact with MAGE-A4 in hepatocellular carcinoma and colorectal cancer, where Gankyrin interaction with MAGE-A4 reduces the oncogenic potential of tumour cells. In this study I aimed to investigate the heterogeneity of GCNIS in relation to disease stage and Sertoli cell development. We also aimed to determine the role of Gankyrin in TGCC cell survival and invasion. The co-expression of early germ cells proteins such as OCT4, LIN28 and PLAP was characterized in GCNIS cells during childhood and adulthood pre-invasive TGCC and in invasive disease characterized by the presence of a testicular tumour. These results show that LIN28 was expressed in 95% of OCT4 GCNIS cells, whereas PLAP expression in GCNIS cells increased as the disease progressed from childhood pre-invasive disease to invasive seminoma (32.3% v 76%; p < 0.05). In contrast there was a reduction in the proportion of MAGE-A4 expressing GCNIS cells with disease progression. The MAGE-A4 expressing population was also less proliferative than the MAGE-A4 negative GCNIS population. The methylation status of GCNIS cells was then investigated. EZH2 a methyltransferase previously reported to be important for TGCC development, was expressed in GCNIS cells at all stages of disease, however the histone 3 modification H3K27me3 (mediated by EZH2) was expressed in a significantly higher percentage of the proliferative OCT4+/MAGE-A4- GCNIS cells compared with the OCT4+/MAGEA4+ population (11.7% v 1.1%; p < 0.01) which could indicate a repressive role for H3K27me3 over MAGE-A4 expression. Next, it was determined whether an association between Sertoli cell maturation status and progression of TGCC could be observed. The maturation status of Sertoli cells was studied using proteins indicative of immature (desmin, cytokeratin, fibronectin and AMH) and mature (vimentin and androgen receptor) Sertoli cells. These studies demonstrated heterogeneity of Sertoli cells maturation in GCNIS-containing tubules. Desmin, fibronectin, AMH and vimentin expression did not show any association with TGCC progression. Cytokeratin was expressed in Sertoli cells of human fetal testis up to second trimester of fetal life, absent in tubules with active spermatogenesis but heterogeneously present in GCNIS, demonstrating that cytokeratin expression is indicative of the presence of GCNIS. Androgen receptor was weakly present in Sertoli cells from human fetal testis and pre-pubertal pre-invasive TGCC testis whereas in GCNIS of adult pre-invasive testis and invasive samples, androgen receptor was abundantly expressed in Sertoli cells of GCNIS-containing tubules. These combined results for cytokeratin and androgen receptor suggest that Sertoli cells from GCNIS-containing tubules, in pre-invasive and invasive TGCC patients are partially differentiated. Gankyrin expression was characterised in fetal germ cells, GCNIS cells and TGCC tissue. In fetal testis nuclear Gankyrin was absent in OCT4+/MAGE-A4- (gonocyte) population whereas it was present in a subpopulation of OCT4-/MAGE-A4+ (spermatogonia) germ cells. In GCNIS cells from TGCC patients nuclear Gankyrin was expressed in 87%, 63.3%, 91.5% and 79% in childhood pre-invasive, adult pre-invasive, seminoma and non-seminoma GCNIS cells respectively. Finally, in seminoma cells, Gankyrin was expressed in the cytoplasm indicating a change in localisation as the GCNIS cells become invasive. We used siRNA to knockdown Gankyrin in NT2 (a TGCC cell line) cells in-vitro and demonstrated a decrease in cell number, suggesting that Gankyrin might play a role in TGCC progression and invasiveness. Gankyrin down-regulation also resulted in an increase in p53 and p21 mRNA level. Given the role of P53 and p21 in cisplatin cytotoxic effect in TGCC we went on to investigate the role of Gankyrin in cisplatin resistance using NT2 cells. We demonstrate that Gankyrin mediated cisplatin resistance through the p53/p21 pathway, upregulating apoptosis rates through BAX and FAS, whilst there was no effect on cell proliferation, cell cycle or cell migration. In conclusion, we have shown that GCNIS cells are heterogeneous and their phenotype can determine their oncogenic potential. We also show that Sertoli cells from GCNIS-containing tubules undergo partial differentiation displaying markers of immature and mature Sertoli cells, with a heterogeneous association of cytokeratin with GCNIS presence. We also demonstrate that the oncogene Gankyrin has a role in NT2 cells survival and cisplatin resistance indicating that manipulation of Gankyrin may have a role in the treatment of TGCC.

Platinum-based therapy is the most often used chemotherapeutic agent to treat advanced cases of head and neck cancers. However, only a small fraction of the patient population responds to cisplatin, with a median survival time of less than a year. Currently, there is a lack of clinically employable molecular characterization of the disease beyond HPV status to classify patients who would respond favorably to platinum-based therapy. In this regard, CD24 expression level appears to be a significant molecular phenotype of cisplatin resistance in laryngeal carcinoma. This study demonstrates that CD24 expression level in HNSCC has a linear relationship with cisplatin resistance, and it affects the transcription of critical apoptotic, stem, and drug resistance genes. The knockdown of the CD24 transcript reduces tumor growth rate and increases the overall cisplatin sensitivity in mice xenograft experiments. A retrospective analysis of a cohort of 25 HNSCC patient tumor samples suggests that CD24-high tumors go on to show an unfavorable response to cisplatin treatment. Overall, based on the strength of further clinical analysis, CD24 presents a strong rationale to be utilized as a predictive indicator to stratify head and neck cancer patients for platinum-based therapy. This study also provides a rationale for using CD24 as a therapeutic adjuvant target along with standard cisplatin therapy in head and neck cancers.

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Introduction Le cancer bronchique est un problème de santé publique en étant la première cause de décès par cancer dans le monde. Il reste de mauvais pronostic avec une résistance au Cisplatine qui est inéluctable dans l’histoire naturelle de la maladie. Nous nous sommes intéressés à l’association du CDDP aux inhibiteurs de la Poly(ADP-ribose) polymérase. Les inhibiteurs pharmacologiques de PARP sont source d’optimisme en oncologie clinique en monothérapie pour des tumeurs déficientes pour une voie de réparation de l’ADN et en association aux cytotoxiques classiques.Matériel et méthodes Nous avons généré 9 clones résistants au CDDP après culture de la lignée A549 dans des faibles doses de CDDP. Deux inhibiteurs pharmacologiques de PARP, CEP8983 (CEP) et PJ34 (PJ), ainsi que des siRNA spécifiques de PARP1 sont utilisés pour l’inhibition de PARP. L’apoptose est mesurée en cytométrie de flux par l’intermédiaire du potentiel membranaire de la mitochondrie DiOC6(3) et la perméabilisation de la membrane plasmique est évaluée par l’iodide de propidium. Le test de clonogénicité permet d’évaluer la capacité des cellules à échapper à la mort et à former une colonie. L’activité métabolique des cellules est mesurée par la mesure de clivage du sel de tetrazolium WST-1. L’immunofluorescence sur cellules fixées a permis d’étudier les dommages de l’ADN (γH2AX), la voie intrinsèque de l’apoptose (l’activation de la caspase 3 et la libération du cytochrome c) et la recombinaison homologue (BRCA1, RAD51). En Western Blot nous avons mesuré l’expression et l’activité de PARP (PAR) ainsi que l’expression d’acteurs de la réparation par excision de base (BER) (XRCC1 and polymérase β). Nous avons développé une méthode de détection de PAR en immunohistochimie sur des tissus inclus en paraffine. Résultats Nous avons trouvé un effet synergique pour l’association du CDDP aux inhibiteurs de PARP in vitro. De façon inattendue nous avons observé que les clones résistants au CDDP développent une addiction à PARP et sont spécifiquement tués par l’inhibition de PARP contrairement à la lignée parentale. Ces clones exhibent une hyperexpression et une hyperactivité de PARP. La réponse aux inhibiteurs de PARP corrèle plus précisément avec l’activation plutôt qu’avec l’expression de PARP, pointant que PAR est un biomarqueur plus précis que PARP. Nous avons observé que l’hyperactivation de PARP accompagne une résistance induite au CDDP et prédispose à une sensibilité aux inhibiteurs de PARP dans d’autres lignées de cancer bronchique (H460 et H1650), de mésothéliome (P31), de cancer de l’ovaire (TOV112D) et de col (HeLa). Dans des expériences in vivo nous avons noté que dans les xénogreffes obtenues à partir de clones résistants au CDDP, l’expression de PAR est stablement retrouvée en immunohistochimie. Ces tumeurs répondaient à l’inhibition de PARP par le PJ en diminuant l’expression de PAR. Les clones résistants au CDDP sensibilisés aux I PARP ont une recombinaison homologue conservée, cependant ont un déficit dans les étapes terminales du BER.Conclusion Nous avons identifié un effet synergique pour l’association des inhibiteurs de PARP au CDDP de des lignées de cancer bronchique. Nous avons observé une dépendance à PARP dans des lignées de cancer bronchique résistantes au CDDP et déficientes pour l’élongation du BER. Nous postulant que PAR est un biomarqueur spécifique de la réponse aux inhibiteurs de PARP. / Introduction Driven by the facts that non small cell lung cancer (NSCLC) is the leading cause of cancer-related morbidity and mortality worldwide and that NSCLC patients often develop resistance against Cisplatin (CDDP)-based therapies, we addressed the question of the combination therapy of CDDP with poly(ADP-ribose) polymerases (PARP) inhibitors. Inhibitors of PARP have raised great expectations for the treatment of a variety of cancers, either as monotherapeutic agent against DNA repair-deficient tumours or combined to DNA-damaging compounds.Material and methods We generated nine CDDP-resistant clones by prolonged exposure to low dose CDDP of the A549 NSCLC parental cell line. Two distinct PARP inhibitors, CEP8983 (CEP) and PJ34 (PJ) as well as PARP1 knockdown with small interfering RNAs (siRNAs) were used for PARP inhibition. Apoptosis was measured by the simultaneous assessment for the loss of the mitochondrial transmembrane potential (m) and the breakdown of the plasma membrane using the m-sensitive fluorochrome DiOC6(3) and the vital dye propidium iodide, respectively. Moreover clonogenic survival was assessed. In vitro assessments of the enzymatic activity of cells were based on the reduction of the colorless tetrazolium salt. Immunofluorescence microscopy determinations were performed with antibodies specific for DNA damage (γH2AX), intrinsic apoptosis (cleaved Caspase-3 and cytochrome c), and homologous recombination (RAD51 and BRCA1). Immunoblotting was assed for PARP1 expression and activity (PAR) and base excision repair (BER) effectors (XRCC1 and polymerase β). We developed an immunohistochemical staining method that specifically detects PAR on paraffin-embedded cell pellets and tissue sections.Results We found that PARP inhibitors and PARP1 siRNAs synergized with CDDP in the killing of NSCLC cells in vitro. Unexpectedly, CDDP-resistant NSCLC cell clones developed addiction to PARP hyperactivation, thereby becoming susceptible to apoptosis induction by PARP inhibition. We showed that these cisplatin-resistant clones, exhibited high PARP protein levels and increased PARP activity, leading to an increased poly-ADP ribosylation of cellular proteins, as compared to their parental, cisplatin-sensitive counterparts. These cisplatin-resistant cells become susceptible to cell death as induced by PARP inhibition, correlating with the hyperactivity of PARP (elevated PAR levels) more accuratly than with the overexpression of PARP. Suggesting that PAR levels may constitute a more accurate biomarker than PARP to predict the sensitivity of cells to PARP inhibition. We expanded the observation that cisplatin resistance causes PARP upregulation and hyperactivation and subsequent sensitization to PARP inhibition to additional five human cancer cell lines including two NSCLC (H1650 and H460), one mesothelioma (P31), one ovarian (TOV112D) and one cervical cancer (HeLa) cell line. To get further insight into this issue, we generated in vivo experiments. Tumors derived from CDDP-resistant cells were characterized by elevated levels of PAR suggesting that PAR levels are preserved during tumor formation. Those PAR-overexpressing tumors responded to the administration of PJ in vivo with a consistent reduction in PAR immunoreactivity. CDDP resistant clones that are specifically killed by PARP inhibitors assessed efficient homologous recombination repair however deficient BER elongation.Conclusion We showed a beneficial effect for the association therapy of PARP inhibitors with CDDP in several NSCLC cell lines. We have identified an addiction to PARP in CDDP resistant cell lines with deficient BER elongation. We postulate that PAR is a specific predictive biomarker for the response to PARP inhibitors.

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Testicular germinal tumors (TGCT) are relatively rare solid tumors in adults. Even so, they affect more than 700 men a year in the Czech Republic, mostly young patients aged 18- 45 years. A large number of patients are curable by a combination of surgery and chemotherapy, yet about 50 men a year in the Czech Republic succumb to this tumor, in the vast majority of cases due to the development of resistance to chemotherapy containing cisplatin. The rare occurrence and high curability are probably the cause of infrequent molecular and clinical studies carried out in these tumors, and our understanding of the biological processes leading to primary tumor development and the development of cisplatin resistance (CDDP) is still limited. At present, no specific molecular markers that could be used as prognostic or predictive factors and improve patient stratification or treatment tailoring are available in TGCT management. In this work, we studied the molecular-genetic background of TGCT development and CDDP resistance at several levels. To comprehensively study the development of cisplatin resistance, we prepared and analyzed CDDP-exposed TGCT cell lines. Long-term exposure to CDDP increased resistance 10-fold in the NCCIT cell line, while no significant resistance was achieved with Tera-2. The...

Cancer is a leading cause of death that affects millions of people across the globe each year. Photodynamic therapy (PDT) is a relatively new treatment approach for cancer in which anticancer drugs are activated by light at an appropriate wavelength to generate highly cytotoxic reactive oxygen species (ROS) and achieve tumor destruction. Compared with conventional chemo- and radiotherapy, PDT can be performed with minimal invasiveness, local targeting and reduced side effects. However, most of the currently available PDT drugs mainly absorb in the visible part of the spectrum, where light penetration depth into human tissues is very limited. Therefore, increasing the treatment depth of PDT has been considered to be an important approach to improve the effectiveness of PDT for treating larger and thicker tumor masses. In this thesis, we present our investigation into the potential of two-photon activated PDT (2-γ PDT), combination therapy of PDT and chemotherapy, and bioluminescence-activated PDT as a means to increase the treatment depth of this modality. In 2-γ PDT, the photosensitizing agents are activated through simultaneous absorption of two photons. This approach allows the use of near-infrared (NIR) light that can penetrate deeper into tissues and thus, has the potential of treating deep-seated tumors and reducing side effects, while the non-linear nature of two-photon excitation (TPE) may improve tumor targeting. We have evaluated the PDT efficacy of a second-generation photosensitizer derived from chlorophyll a, pyropheophorbide a methyl ester (MPPa), through both one- and two-photon activation. We observed that MPPa had high one-photon (1-γ PDT efficacy against both cisplatin-sensitive human cervical (HeLa) and cisplatin-resistant human lung (A549) and ovarian (NIH:OVCAR-3) cancer cells when activated by femtosecond (fs) laser pulses at 674 nm. At a low light dose of 0.06 J cm-2, the MPPa concentration required to produce a 50% cell killing effect (IC50) was determined to be 5.3 ± 0.3, 3.4 ± 0.3 and 3.6 ± 0.4 μM in HeLa, A549 and NIH:OVCAR-3 cells, respectively. More significantly, we also found that MPPa could be effectively activated at the optimal tissue-penetrating wavelength of 800 nm through TPE. At a light dose of 886 J cm-2, where no measurable photodamage was observed in the absence of MPPa, the IC50 values were measured to be 4.1 ± 0.3, 9.6 ± 1.0 and 1.6 ± 0.3 μM in HeLa, A549 and NIH:OVCAR-3 cells, respectively. We obtained corresponding LD50 (the light dose required to produce a 50% killing effect) values of 576 ± 13, 478 ± 18 and 360 ± 16 J cm-2 for 10 μM MPPa, which were approximately 3-5 times lower than the published 2-γ LD50 of Visudyne® and 20-30 times lower than that of Photofrin®. These results indicate that MPPa may serve as a photosensitizer for both 1- and 2-γ activated PDT treatment of difficult-to-treat tumors by conventional therapies. Indocyanine green (ICG), a dye having an absorption maximum near 800 nm, has been considered to be a potential NIR PDT agent. However, the PDT efficacy of ICG has been found to be very limited probably due to the low yield of cytotoxic ROS. In the present work, we have evaluated the combination effects of ICG-mediated PDT with conventional chemotherapy mediated by two types of chemotherapeutic drugs, namely the type II topoisomerase (TOPII) poisons etoposide (VP-16)/teniposide (VM-26) and the platinum-based drugs cisplatin (CDDP)/oxaliplatin (OXP). Synergistic enhancement of cytotoxicity and increased yields of DNA double strand breaks (DSBs) were observed in HeLa, A549 and NIH:OVCAR-3 cancer cells treated with the combination of ICG-PDT and VP-16. The presence of VP-16 during the laser irradiation process was found to be critical for producing a synergistic effect. An electron-transfer-based mechanism, in which ICG could increase the yield of highly cytotoxic VP-16 metabolites, was proposed for the observed synergistic effects, although direct spectroscopic detection of the reaction products was found to be very challenging. Moreover, we observed a much lower degree of synergy in the human normal fibroblast GM05757 cells than that in the three cancer cell lines investigated. Synergistic effects were also observed in A549 cells treated with the combination of ICG-PDT and VM-26 (i.e. an analog of VP-16). Furthermore, the combination of low-dose CDDP/OXP and ICG-PDT was demonstrated to produce an additive or synergistic effect in selected cancer cell lines. These preliminary results suggest that the combination of ICG-PDT with VP-16/VM-26 or CDDP/OXP chemotherapy may offer the advantages of enhancing the therapeutic effectiveness of ICG-PDT and lowering the side effects associated with the chemotherapeutic drugs. Bioluminescence, the generation of light in living organisms through chemical reactions, has been explored as an internal light source for PDT in recent years. This approach, in principle, does not suffer from the limited tissue penetration depth of light. In the present project, we have evaluated the effectiveness of luminol bioluminescence in activating the porphyrin photosensitizers meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TPPS4) and Fe(III) meso-tetra(4-sulfonatophenyl)porphine chloride (FeTPPS). The combination treatment induced significant killing of HeLa cells, while additive effects were observed in two normal human fibroblast cell lines (GM05757 and MRC-5). Our observations indicate that bioluminescence of luminol may generate sufficient light for intracellular activation of PDT sensitizers. Furthermore, the combination treatment may have intrinsic selectivity towards cancerous tissues. In summary, we have demonstrated effective killing of cancer cells by MPPa-mediated 1- and 2-γ PDT, combination of ICG-PDT and VP-16/VM-26 or CDDP/OXP chemotherapy, and bioluminescence of luminol activated PDT mediated by TPPS4/FeTPPS. These positive preliminary results indicate that all these three approaches have the potential of increasing the treatment depth of PDT and facilitating the development of more effective PDT treatment strategies.