Colorectal cancer is the second leading cause of cancer-related deaths in the United States and the third most common cancer in men and women. Vitamin E is a lipid soluble antioxidant that exists as eight structurally different isoforms of tocopherols and tocotrienols. Recent experimental, and molecular studies suggest that γ-tocotrienol (GT3) may be a more potent cancer-preventive form of vitamin E. 15-lipoxygenase-1 (15-LOX-1) and its product 13-S-hydroxyoctadecadienoic acid (13-S-HODE) are decreased in colon cancer cells. 15 LOX-1 is considered a tumor suppressor gene in colon carcinogenesis. Non-steroidal anti-inflammatory drug (NSAID)-induced 15-LOX-1 expression is critical to aspirin and NSAID-induced apoptosis in colorectal cancer cells. HCT-116 is a microsatellite-instability (MSI) colon cancer cell line. MSI is a marker of chemo-resistance but is associated with improved survival as compared to microsatellite-stable (MSS) colon cancers. The effects of GT3 on cytotoxicity and 15 LOX-1 expression was studied on the human colon cancer cell line HCT-116. HCT-116 colon cancer cell lines were cultured in DMEM media and dosed with increasing concentrations of GT3 (20µM-50µM). Cytotoxicity of the drugs was studied using Cell Titer Glo and MTS assays 24 hours after dosing. Cells were then plated in 6-well plates and grown for 24 hours. Cells were then dosed with 2 mL of GT3 at 20 uM at the respective time periods (2h, 4h, 6h, 12h, 16h, 24h) and lysates were harvested. Gel electrophoresis was run according to BCA protein assay from the time-dependent lysates and blots were tagged with a rabbit 15-lox antibody. Ongoing experiments include RNA PCR. RNA is being isolated at 2, 4, 6 and 12 hours. The RNA as reversed transcribed using a 15 lox 1 primer and that cDNA is being quantified using Quantitative PCR. GT3 induced cytotoxicity in MTS assay and Cell Titer Glo assay when added to HCT-116 cell line. 15 LOX 1 protein expression was found to be up-regulated in the colon cancer cell line HCT-116 when GT3 was added at 12h, 16h and 24h with the maximum expression at 16 hours. Chemotherapeutic drugs can have significant side effects. Understanding the role of GT3 on colon cancer cell lines could lead to the development of novel drugs to supplement current chemotherapy regimens and allow for lower doses of chemotherapeutic agents. Modulation of 15-LOX-1 suggests that GT3 may induce apoptosis through induction of the lipoxygenase pathway. Further experiments are under way to study the mechanism of action of GT3 on the 15 LOX-1 pathway. Since HCT-116 is a MSI- colon cancer cell line, effects of GT3 on MSS- colon cancer cell lines will also be studied.

O Glioblastoma (GBM) é um astrocitoma grau IV, representando o glioma de maior malignidade e o tumor cerebral primário mais frequente em humanos. A terapia indicada para o GBM é a ressecção cirúrgica, quimioterapia e radioterapia, todas com baixíssima eficiência devido a agressividade e as características do GBM. Consequentemente, a sobrevida dos pacientes indicados aos tratamentos convencionais é pouco mais de um ano. A inflamação é, sabidamente, uma das características que participa de modo decisivo do desenvolvimento tumoral, incluindo do GBM, e as relações entre mediadores inflamatórios e câncer são alvos de pesquisa nos últimos anos. Diversos estudos apontam um papel da via dos eicosanoides na modulação de processos patológicos envolvidos na inflamação e no câncer. Os eicosanoides são mediadores lipídicos bioativos, envolvidos em diversos processos fisiológicos e patológicos, em especial os associados à resposta inflamatória. As principais vias de produção de eicosanoides (ciclooxigenases, lipoxigenases, e citocromo P450), assim como seus produtos, são frequentemente alterados em diversos tipos de tumor, associados ao crescimento e a progressão tumoral. Contudo, o perfil dessas vias é consideravelmente pouco compreendido em GBM. O objetivo deste estudo foi analisar in vitro o perfil e o papel das três vias de eicosanoides e seus produtos (eicosanoides ou não) nas linhagens de GBM (U251-MG, U87-MG, A172, T98G e U138-MG), modulando a atividade de enzimas chaves com drogas especificas para, então, analisar parâmetros de proliferação, migração e morte celular. Nossos resultados mostram, em todas as linhagens analisadas, um perfil heterogêneo das enzimas e receptores chaves das três vias. O perfil lipídico evidencia a produção de 13-HODE, produto de 15-lipoxigenase-1 em todas as linhagens, bem como ausência de leucotrienos e 5-HETE do eixo de 5-lipoxigenase. Os inibidores farmacológicos para 15-LOX e 12-LOX/15-LOX foram capazes de reduzir o crescimento, modular o ciclo celular e a migração celular das linhagens U251-MG, U87-MG e A172. O mesmo é visto com a inibição de mPGES-1. A inibição de 5-LOX por outro lado não afetou nenhum parâmetro nas mesmas linhagens. Todos os resultados apontam, portanto, para um papel do eixo de 15-LOX e COX no crescimento e na migração das células de GBM humano. / Glioblastoma (GBM) is a grade IV astrocytoma, the most malignant and the most frequent primary brain tumour in humans. Standard therapies for treating GBM are surgical resection, chemotherapy and radiotherapy, all with low efficiency due to GBM aggressiveness and characteristics. Therefore, patient survival after conventional treatments is about one year. Inflammation is one of the main characteristics that plays a decisive role in tumour development, including in GBM. The relationships between inflammatory mediators and cancer have been the subject of research in recent years. Several studies point to the eicosanoid pathways as modulators of pathological processes between inflammation and cancer. Eicosanoids are bioactive lipid mediators, involved in various physiological and pathological processes, especially those associated with the inflammatory response. The major eicosanoid pathways (cyclooxygenases, lipoxygenases, and cytochrome P450) as well as their products, are frequently altered in several tumours, associated with tumour growth and progression. However, the profile of these pathways is poorly understood in GBM. The objective of this study was to analyse, in vitro, the profile and role of eicosanoid pathways and their products (eicosanoids or not) in GBM cell lines (U251-MG, U87-MG, A172, T98G and U138-MG), modulating the key enzymes with inhibitors, analysing proliferation, migration and cell death. Our results show, in all analysed cell lines, a heterogeneous profile for the key enzymes and receptors of the three pathways. The lipid profile shows the production of 13-HODE, a product of 15-lipoxygenase-1, as well the absence of leukotrienes and 5-HETE of the 5-lipoxygenase axis. The pharmacological inhibitors for 15-LOX and 12-LOX / 15-LOX led to changes in cell cycle, reduced growth, reduced migration of U251-MG, U87-MG and A172 cell lines. The same was seen with the inhibition of mPGES-1. Inhibition of 5-LOX, on the other hand, did not affect any of these parameters in the same cell lines. All results, therefore, point to an important role of 15-LOX and COX pathways in the growth and migration of human GBM cells.