Lebanese Plants and Plant-Derived Compounds Against Colon Cancer

Colorectal cancer (CRC) is a major health concern and demands long-term efforts in developing strategies for screening and prevention. CRC has become a preventable disease as a consequence of a better understanding of colorectal carcinogenesis. However, current therapy is unsatisfactory and necessitates the exploration of other approaches for the prevention and treatment of cancer. Plant based products have been recognized as preventive with regard to the development of colon cancer. Therefore, the potential chemopreventive use and mechanism of action of Lebanese natural product were evaluated. Towards this aim the antitumor activity of Onopordum cynarocephalum and Centaurea ainetensis has been studied using in vitro and in vivo models. In vitro, both crude extracts were non cytotoxic to normal intestinal cells and inhibited the proliferation of colon cancer cells in a dose-dependent manner. In vivo, both crude extracts reduced the number of tumors by an average of 65% at weeks 20 (adenomas stage) and 30 (adenocarcinomas stage). The activity of the C. ainetensis extract was attributed to Salograviolide A, a guaianolide-type sesquiterpene lactone, which was isolated and identified through bio-guided fractionation. The mechanism of action of thymoquinone (TQ), the active component of Nigella sativa, was established in colon cancer cells using in vitro models. By the use of N-acetyl cysteine, a radical scavenger, the direct involvement of reactive oxygen species in TQ-induced apoptotic cells was established. The analytical detection of TQ from spiked serum and its protein binding were evaluated. The average recovery of TQ from spiked serum subjected to several extraction procedures was 2.5% proving the inability of conventional methods to analyze TQ from serum. This has been explained by the extensive binding (>98%) of TQ to serum and major serum components such as bovine serum albumin (BSA) and alpha-1-acid glycoprotein (AGP). Using mass spectrometry analysis, TQ was confirmed to bind covalently to the free cysteine in position 34 and 147 of the amino acid sequence of BSA and AGP, respectively. The results of this work put at the disposal for future development new plants with anti-cancer activities and enhance the understanding of the pharmaceutical properties of TQ, a prerequisite for its future clinical development.

Neuro- and immunotoxic effects of fumonisin B1 in cells

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other agricultural products. Fusarium species can also be found in moisture-damaged buildings, and therefore there may also be human exposure to Fusarium mycotoxins, including FB1. FB1 affects the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase. It is neuro-, hepato- and nephrotoxic, and it is classified as possibly carcinogenic to humans. This study aimed to clarify the mechanisms behind FB1-induced neuro- and immunotoxicity. Four neural and glial cell lines of human, rat and mouse origin were exposed to graded doses of FB1 and the effects on the production of reactive oxygen species, lipid peroxidation, intracellular glutathione levels, cell viability and apoptosis were investigated. Furthermore, the effects of FB1, alone or together with lipopolysaccharide (LPS), on the mRNA and protein expression levels of different cytokines and chemokines were studied in human dendritic cells (DC). FB1 induced oxidative stress and cell death in all cell lines studied. Generally, the effects were only seen after prolonged exposure at 10 and 100 µM of FB1. Signs of apoptosis were also seen in all four cell lines. The sensitivities of the cell lines used in this study towards FB1 may be classified as human U-118MG glioblastoma > mouse GT1-7 hypothalamic > rat C6 glioblastoma > human SH-SY5Y neuroblastoma cells. When comparing cell lines of human origin, it can be concluded that glial cells seem to be more sensitive towards FB1 toxicity than those of neural origin. After exposure to FB1, significantly increased levels of the cytokine interferon-γ (IFNγ) were detected in human DC. This observation was further confirmed by FB1-induced levels of the chemokine CXCL9, which is known to be regulated by IFNγ. During co-exposure of DC to both LPS and FB1, significant inhibitions of the LPS-induced levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β, and their regulatory chemokines CCL3 and CCL5 were observed. FB1 can thus affect immune responses in DC, and therefore, it is rather likely that it also affects other types of cells participating in the immune defence system. When evaluating the toxicity potential of FB1, it is important to consider the effects on different cell types and cell-cell interactions. The results of this study represent new information, especially about the mechanisms behind FB1-induced oxidative stress, apoptosis and immunotoxicity, as well as the varying sensitivities of different cell types towards FB1.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.