No description available.

Chemotherapy is often administered once every three weeks to allow repopulation of essential normal tissues such as the bone marrow. Repopulation of surviving tumour cells can also occur between courses of chemotherapy and can decrease the efficacy of anticancer treatment. This thesis aims to characterize repopulation, to study the effect of targeted cytostatic agents to inhibit repopulation, and to determine the optimal scheduling of chemotherapy and molecular targeted treatment. The distribution of proliferating and apoptotic cells in human squamous cell carcinoma (A431) xenografts was studied following chemotherapy using fluorescence immunohistochemistry. There was an initial decrease in cell proliferation and in the total functional blood vessels, and an increase in apoptosis observed following treatment with paclitaxel chemotherapy. A rebound in cell proliferation occurred approximately 12 days following treatment, which corresponded with a rebound in vascular perfusion. The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation between courses of chemotherapy was determined using EGFR-overexpressing A431 cells and xenografts. Furthermore, concurrent and sequential schedules of combined chemotherapy and molecular targeted treatment were compared. Gefitinib inhibited the repopulation of A431 cells in culture when administered sequentially between chemotherapy; sequential treatment was more efficacious than concurrent treatment probably because concomitant scheduling rendered quiescent cells less responsive to chemotherapy. However, in vivo studies using chemotherapy in combination with gefitinib or temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, showed that concurrent scheduling of combined treatment was more effective at delaying regrowth of xenografts than sequential treatment; this was likely due to dominant effects on the tumour microenvironment. The work completed in this thesis has shown that repopulation occurs in A431 xenografts following paclitaxel treatment, and these changes are associated with changes in the tumour vasculature. Repopulation of A431 cells was inhibited by gefitinib administered sequentially with paclitaxel. However, studies in mice showed better inhibitory effects when chemotherapy was given concomitantly with cytostatic agents such as gefitinib or temsirolimus. Our in vivo data highlight the importance of characterizing changes in the tumour microenvironment when determining optimal scheduling of chemotherapy and molecular targeted treatment.

Chemotherapy is often administered once every three weeks to allow repopulation of essential normal tissues such as the bone marrow. Repopulation of surviving tumour cells can also occur between courses of chemotherapy and can decrease the efficacy of anticancer treatment. This thesis aims to characterize repopulation, to study the effect of targeted cytostatic agents to inhibit repopulation, and to determine the optimal scheduling of chemotherapy and molecular targeted treatment. The distribution of proliferating and apoptotic cells in human squamous cell carcinoma (A431) xenografts was studied following chemotherapy using fluorescence immunohistochemistry. There was an initial decrease in cell proliferation and in the total functional blood vessels, and an increase in apoptosis observed following treatment with paclitaxel chemotherapy. A rebound in cell proliferation occurred approximately 12 days following treatment, which corresponded with a rebound in vascular perfusion. The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation between courses of chemotherapy was determined using EGFR-overexpressing A431 cells and xenografts. Furthermore, concurrent and sequential schedules of combined chemotherapy and molecular targeted treatment were compared. Gefitinib inhibited the repopulation of A431 cells in culture when administered sequentially between chemotherapy; sequential treatment was more efficacious than concurrent treatment probably because concomitant scheduling rendered quiescent cells less responsive to chemotherapy. However, in vivo studies using chemotherapy in combination with gefitinib or temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, showed that concurrent scheduling of combined treatment was more effective at delaying regrowth of xenografts than sequential treatment; this was likely due to dominant effects on the tumour microenvironment. The work completed in this thesis has shown that repopulation occurs in A431 xenografts following paclitaxel treatment, and these changes are associated with changes in the tumour vasculature. Repopulation of A431 cells was inhibited by gefitinib administered sequentially with paclitaxel. However, studies in mice showed better inhibitory effects when chemotherapy was given concomitantly with cytostatic agents such as gefitinib or temsirolimus. Our in vivo data highlight the importance of characterizing changes in the tumour microenvironment when determining optimal scheduling of chemotherapy and molecular targeted treatment.

No description available.

Anti-cancer drugs gain access to solid tumors via the blood, and must penetrate tissue to reach all viable cancer cells. This thesis aims to compare the distribution of anticancer drugs in normal tissues and tumours, to examine whether drug distribution is modifiable and quantifiable in solid tumours, and to determine whether extracellular drug distribution can be improved by modifying intracellular drug distribution. The time-dependent spatial distribution of three anticancer drugs, doxorubicin, mitoxantrone and topotecan, were studied in normal tissues and tumours. Ten minutes after drug administration, there was fairly uniform distribution in the heart, kidney and liver whereas drug distribution within tumours was limited to perivascular regions. Doxorubicin distribution in P-glycoprotein (PgP) over-expressing tumours was compared to that in wild-type tumours and changes in distribution were evaluated with the use of PgP inhibitors. There was better doxorubicin distribution in PgP-over-expressing tumours compared to wild-type tumours, and pretreatment of PgP-over-expressing tumours with PgP inhibitors decreased doxorubicin distribution. These data suggest that reduced uptake of drug into cells may enhance extracellular drug distribution, and the dual effects of PgP inhibitors (increased drug uptake in proximal cells, but poorer drug distribution) may explain, in part, why these agents have not provided clinical benefit. The effect of the proton pump inhibitor pantoprozole on intracellular and extracellular drug distribution was determined. Pantoprazole increased endosomal pH in cells, leading to less sequestration of doxorubicin within them, and increased the toxicity of doxorubicin for cultured cells. In wild-type MCF7 tumours, pretreatment with pantoprazole enhanced doxorubicin distribution and tumour growth delay without apparent increase in toxicity. These studies have led to initiation of a phase I clinical trial of pantoprazole and doxorubicin for patients with solid tumours. The work completed in this thesis demonstrates that drug distribution can be modified and that these changes can be quantified, and may correlate with improved anti-tumour effects. Improving drug distribution through the use of proton pump inhibitors may be an effective strategy to improve chemotherapeutic efficacy.

Anti-cancer drugs gain access to solid tumors via the blood, and must penetrate tissue to reach all viable cancer cells. This thesis aims to compare the distribution of anticancer drugs in normal tissues and tumours, to examine whether drug distribution is modifiable and quantifiable in solid tumours, and to determine whether extracellular drug distribution can be improved by modifying intracellular drug distribution. The time-dependent spatial distribution of three anticancer drugs, doxorubicin, mitoxantrone and topotecan, were studied in normal tissues and tumours. Ten minutes after drug administration, there was fairly uniform distribution in the heart, kidney and liver whereas drug distribution within tumours was limited to perivascular regions. Doxorubicin distribution in P-glycoprotein (PgP) over-expressing tumours was compared to that in wild-type tumours and changes in distribution were evaluated with the use of PgP inhibitors. There was better doxorubicin distribution in PgP-over-expressing tumours compared to wild-type tumours, and pretreatment of PgP-over-expressing tumours with PgP inhibitors decreased doxorubicin distribution. These data suggest that reduced uptake of drug into cells may enhance extracellular drug distribution, and the dual effects of PgP inhibitors (increased drug uptake in proximal cells, but poorer drug distribution) may explain, in part, why these agents have not provided clinical benefit. The effect of the proton pump inhibitor pantoprozole on intracellular and extracellular drug distribution was determined. Pantoprazole increased endosomal pH in cells, leading to less sequestration of doxorubicin within them, and increased the toxicity of doxorubicin for cultured cells. In wild-type MCF7 tumours, pretreatment with pantoprazole enhanced doxorubicin distribution and tumour growth delay without apparent increase in toxicity. These studies have led to initiation of a phase I clinical trial of pantoprazole and doxorubicin for patients with solid tumours. The work completed in this thesis demonstrates that drug distribution can be modified and that these changes can be quantified, and may correlate with improved anti-tumour effects. Improving drug distribution through the use of proton pump inhibitors may be an effective strategy to improve chemotherapeutic efficacy.

HOXA7 belongs to a family of homeobox transcription factors that are master regulators of cell differentiation, morphogenesis during embryonic development and cell proliferation. Dysregulation and non-nuclear localization of these proteins play a role in a large number of solid tumours, with reports of significant upregulation of HOXA7 in oral dysplasia. It is unclear whether HOXA7 induction in solid tumours is causative or if it is a result of oncogenic changes. In this thesis we studied its effect on cell differentiation, growth, stemness, cell migration, EMT and cell senescence. The main hypothesis was that HOXA7 regulated keratinocyte differentiation through the regulation of activator protein 1 (AP-1), a keratinocyte specific activator of differentiation. We also hypothesised that HOXA7 increased the proliferation rate in keratinocytes. In an AP-1 reporter assay in HEK293 cells, HOXA7 was shown to decrease AP-1 activity significantly. The inactivation of AP-1 was not due to inactivation of PKC, as HOXA7 did not interfere with the activation of the kinases in HEK293. More specifically, we reported a very significant repression of c-Jun and JunD promoter activity in the presence of ectopic HOXA7 in HEK293 cells. We further showed that this mechanism might also be applicable in keratinocytes, as HOXA7 inhibited the transcription of AP-1 subunits of both the Jun and Fos family in skin keratinocytes. Furthermore, we showed transcriptional repression of four differentiation markers and a downregulation of K1 and FLG protein in transduced NEB-1 monolayers as well as K1 suppression in HaCaT cells. The organotypic cultures revealed a downregulation of K1, K10, and filaggrin in stratified HaCaT cells by HOXA7. There was however no downregulation in oral keratinocytes. These observations taken together suggested that HOXA7 repressed the synthesis of AP-1 units in skin keratinocytes, which would have resulted in reduced quantities of AP-1 and therefore lower activity. Contrary to previous reports, we observed no positive involvement of HOXA7 in keratinocyte proliferation, EMT or migration. There was however an indication of cell-type specific MET and induced cell senescence. Based on our results we propose a cell-type specific role of HOXA7 as an antagonist of AP-1 transcription in skin keratinocytes, and a possible direct binding of HOXA7 to c-Jun and JunD promoters.

Within solid tumours, a microenvironment exists that causes resistance to chemotherapy. New drugs that target cells within this microenvironment are required, the first step in this process being the identification of new targets. The aim of this thesis was to characterise changes in the transcriptome and proteome within specific regions of multicell-tumour spheroids (MCTS), an experimental model that mimics many of the features of the tumour microenvironment. HT29 MCTS were separated by sequential trypsinisation into 3 main regions; the outer surface layer (SL) the peri-necroric region (PN) and the necrotic core (NC). Using an iTRAQ quantitative proteomics approach, the proteome of the different MCTS regions was investigated. A 2 dimensional separation approach using Agilent's OffGel system and RP-nano HPLC was incorporated prior to MS analysis. MS analysis was done using both MALDI-TOF-TOF (Bruker Ultraflex II) and ESI-Q-TOF (Agilent 6530 QTOF LC/MS) instruments. Gene expression profiles of the different MCTS were investigated and compared using Agilent's one-color oligonucleotide based microarrays. Transcriptomic and proteomic analysis identified several key differences in the proteins involved in cell metabolism between the SL and PN/NC regions. Similar metabolic changes were also noted between autophagic and normal monolayer cells. Many highlighted proteins represented established cancer associated proteins. Interestingly, a number of proteins were highlighted which have no previous association with cancer and may upon further validation, provide attractive leads for therapeutic intervention.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Dimitrios Vagiannis Supervisor RNDr. Jakub Hofman, Ph.D. Title of Doctoral Thesis Study on the role of pharmacokinetic mechanisms of drug resistance in new anticancer drugs with focus on solid tumors Cancer chemotherapy is an important tool for the cure of cancer. Although the development of new anticancer drugs has been rapidly progressing, the phenomenon of multidrug resistance (MDR) continues to be a key issue leading to therapy failure in oncological patients. MDR is based on pharmacodynamic as well as pharmacokinetic mechanisms. Pharmacokinetic MDR includes drug efflux transporters and biotransformation enzymes that decrease the amount of (active form of) a drug in tumors. While the MDR role of transporters has been well understood, the participation of drug metabolizing enzymes is still unclear. This thesis investigates the role of cytochromes P450 (CYPs) in cytostatic resistance. Furthermore, it focuses on the modulation of pharmacokinetic MDR using pharmacokinetic drug-drug interactions of new targeted antitumor drugs. Finally, it aims to confirm the in vitro findings in ex vivo patient-derived tumor explants. In our latest publication, we demonstrate the significant role of...

Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)