Molecular aspects

Prostate cancer is one of the leading causes of death in men aged 40-55. Genistein isoflavone (4', 5', 7-trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti-tumor activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy drug, primarily used for secondary treatment of ovarian,cervical and small cell lung cancers. This study was to demonstrate the potential anticancer activities and synergy of topotecan-genistein combination in LNCaP prostate cancer cells. The potential efficacy and mechanism of topotecan/genistein-induced cell death was investigated... Results: The overall data indicated that i) both genistein and topotecan induce cellular death in LNCaP cells, ii) topotecan-genistein combination was significantly more efficacious in reducing LNCaP cell viabiligy compared to either genistein or topotecan alone, iii) in all cases, cell death was primarily through apoptosis, via the activation of the intrinsic pathway, iv) ROS levels were increased and VEGF expression was diminished significantly with the topotecan-genistein combination treatment, v) genetic analysis of topotecan-genistein treatment groups showed changes in genetic expression levels in pathway specific apoptotic genes.... Conclusion: Treatments involving topotecan-genistein combination may prove to be an attractive alternative phytotherapy of adjuvant therapy for prostate cancer.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed.

We attempted to understand the molecular regulators that impact inflammation using a rat model of human sensation-seeking/risk-taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress. We found that HRs have a pro-inflammatory phenotype as indicated by increased protein expression of the inflammatory cytokine TNF-(Sa(B. Furthermore, we found that HRs have a lower gene expression of the glucocorticoid receptor and histone deacetylase 2 which are known to play an immunosuppressive role. Autophagy (macroautophagy) is a homeostatic process needed for cell maintenance, growth and proliferation and known to assist in tumor survival. FYVE and coiled-coil domain containing 1 (FYCO1) is a novel protein implicated to assist in the plus-end directed trafficking and fusion of autophagosomes. In these studies, we show that FYCO1 gene expression among human breast cell lines of varying degrees of malignancy.

Drosophila melanogaster encounter periods of increased temperature or decreased oxygen in its native environment. One consequence of these environmental stresses is increased production of reactive oxygen species that damage major molecules within cells. Another consequence is that flies fall into a protective coma where biological functions are minimized to conserve energy expenditures. This biological phenomenon is called spreading depression. The overarching aim of this project is to determine if methionine sulfoxide reductases affect entrance or exit from the protective coma induced by acute thermal stress. The data revealed that complete deficiency of Msr in young flies causes a faster induction of the coma. In both young and old flies, Msr does not affect average recovery time but does affect the pattern of recovery from coma. Entrance into the coma is age dependent with young flies maintaining activity longer than before entering into the coma as compared to old flies.

Huntington's Disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the Huntingtin gene IT15. In this study we demonstrated that Bcl-2 interacting mediator of cell death Extra Long (BimEL) protein expression was significantly increased in cells expressing mutant Huntingtin (mHtt). Moreover, striatal BimEL expression remained high in an R6/2 HD mouse model throughout the disease progression. Utilizing novel BimEL phospho-mutants we demonstrated the phosphorylation of Ser65 to be important for the stabilization of BimEL. We provided evidence that impaired proteasome function, increased JNK activity and reduced striatal BDNF lead to changes in the phosphorylation of BimEL, thereby promoting its stabilization specifically within the striatum of R6/2 mice. Furthermore, knocking down BimEL expression prevented mHtt-induced cell death in a HD cell culture. Taken together, these findings suggest that BimEL may contribute to the selective neurodegeneration and pathogenesis of HD.

The enzyme Methionine sulfoxide reductase A (MsrA) repairs oxidized proteins, and may act as a scavenger of reactive oxygen species (ROS), making it a potential therapeutic target for age-related neurodegenerative diseases. The anoxia-tolerant turtle offers a unique model to observe the effects of oxidative stress on a system that maintains neuronal function following anoxia and reoxygenation, and that ages without senescence. MsrA is present in both the mitochondria and cytosol, with protein levels increasing respectively 3- and 4-fold over 4 hours of anoxia, and remaining 2-fold higher than basal upon reoxygenation. MsrA was knocked down in neuronally-enriched cell cultures via RNAi transfection. Propidium iodide staining showed no significant cell death during anoxia, but this increased 7-fold upon reoxygenation, suggesting a role for MsrA in ROS suppression during reperfusion. This is the first report in any system of MsrA transcript and protein levels being regulated by oxygen levels.

Prostate cancer is the most common form of non-skin cancer and the second leading cause of cancer deaths within the United States. The five year survival rate has increased from 69% to 99% over the last 25 years for the local and regional disease, but has remained fairly low (approximately 34%) for the advanced disease. Therefore, current research is aimed at finding complementary or alternative treatments that will specifically target components of the signal transduction, cell-cycle and apoptosis pathways to induce cell death, with little or no toxic side effects to the patient. In this study we investigated the effect of genistein on expression levels of genes involved in these pathways. Genistein is a (4 , 5 , 7-trihydroxyisoflavone) is a major isoflavone constituent of soy that has been shown to inhibit growth proliferation and induce apoptosis in cancer cells. The mechanism of genistein-induced cell death and potential molecular targets for genistein in LNCaP prostate cancer c ells was investigated using several techniques. The chemosensitivity of genistein towards the prostate cancer cells was investigated using the ATP and MTS assays and apoptosis induction was determined using apoptosis and caspase assays. Several molecular targets were also identified using cDNA microarray and RT-PCR analysis. Our results revealed that genistein induces cell death in a time and dose-dependent manner and regulates expression levels of several genes involved in carcinogenesis and immunogenicity. Several cell cycle genes were down-regulated, including the mitotic kinesins, cyclins and cyclin dependent kinases, indicating that genistein is able to halt cell cycle progression through the regulation of genes involved in this process.

Cancer is the second leading cause of death in the western world. In order to treat various types of cancer, platinum-based drugs are most widely employed as metal-containing chemotherapeutic agents. However, their clinical usage is hindered by toxic side effects, and by the emergence of drug resistance. Our focus was to replace platinum with less toxic metal like tin which can give better alternatives for cancer treatment. The major aim of our study was to synthesize novel organotin polyethers (Sn-O) which can be used to combat cancer. Preliminary results from our laboratory using organotin polyethers, that were synthesized by varying the structure of diols showed growth inhibition in Balb-3T3 cells. This study directly led us to hypothesize the two structural windows, first by changing the distance between diol and second, by presence of unsaturation in diols, the biological activity of organotin polyethers (Sn-O) can be enhanced significantly. Different series of polymeric compounds were synthesized based upon these two structural windows and the formation of products was validated using standard techniques like infrared spectroscopy (IR), light scattering photometer, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nuclear magnetic resonance (NMR). The synthesized polymers arrested the growth of cancer cell lines including bone, prostate, colon, breast, pancreas and lung cancer derived cell lines in vitro. In number of instances where chemotherapeutic index values of two and greater were found that these polymers are significantly more active against cancer cells than non-cancerous cells in culture.

Microarray expression data which contains the expression levels of a large number of simultaneously observed genes have been used in many scientific research and clinical studies. Due to its high dimensionalities, selecting a small number of genes has shown to be beneficial for many tasks such as building prediction models from the microarray expression data or gene regulatory network discovery. Traditional gene selection methods, however, fail to take the class distribution into the selection process. In biomedical science, it is very common to have microarray expression data which is severely biased with one class of examples (e.g., diseased samples) significantly less than other classes (e.g., normal samples). These sample sets with biased distributions require special attention from researchers for identification of genes responsible for a particular disease. In this thesis, we propose three filtering techniques, Higher Weight ReliefF, ReliefF with Differential Minority Repeat and ReliefF with Balanced Minority Repeat to identify genes responsible for fatal diseases from biased microarray expression data. Our solutions are evaluated on five well-known microarray datasets, Colon, Central Nervous System, DLBCL Tumor, Lymphoma and ECML Pancreas. Experimental comparisons with the traditional ReliefF filtering method demonstrate the effectiveness of the proposed methods in selecting informative genes from microarray expression data with biased sample distributions.