Browsing by Subject "Melanoma"
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Item Engineering a novel human methionine degrading enzyme as a broadly effective cancer therapeutic(2014-08) Paley, Olga M.; Georgiou, George; Iverson, Brent; Alper, Hal S; Maynard, Jennifer; Johnson, Kenneth AMany cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic.Item Insulin-Mediated Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cell Cultures(Texas Tech University, 1984-05) Ehlers, Susan ElizabethInsulin lowers basal levels of tyrosinase activity and inhibits the MSH-stimulated rise in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. These cultures are very sensitive to insulin. A concentration of insulin as low as 5 x 10 M insulin produces optimum inhibition. At maximum inhibition, tyrosinase activity is reduced to approximately 50% of control levels. Insulin inhibits cellular proliferation in melanoma cells; however, inhibition of tyrosinase activity precedes this effect. Insulin also inhibits the (Bu)2 cAMP and theophylline stimulated rise in enzyme activity. This finding suggests that insulin exerts its effects at a site distal to cAMP production. Insulin, in fact, does not lower cAMP levels in melanoma cells, nor does it alter the MSH-stimulated rise of cAMP. The inhibitory effect of insulin on tyrosinase activity could not be mimicked by either (Bu)2 cGMP or 8-bromo-cGMP, suggesting that insulin does not exert its effects by altering cellular levels of this nucleotide. Insulin decreases the incorporation of [3H]-leucine into trichloracetic acid insoluble material by 50%, an inhibition which corresponds well with the observed level of reduction of tyrosinase activity. This finding suggests that the inhibition of tyrosinase activity may be caused by a general reduction in protein synthesis due to insulin treatment.Item Obesity promotes B16BL6 melanoma cell invasiveness and Snai1 expression(2012-05) Kushiro, Kyoko; Nuñez, Nomelí P.; Hursting, Stephen D.Malignant melanoma is cancer arising from melanocytes that have acquired the ability to metastasize and colonize secondary organs such as the lungs, liver, and brain. According to the Melanoma Research Foundation, malignant melanoma is the most rapidly increasing type of cancer with an annual incidence increase of ~ 4% despite the therapeutic and medical breakthroughs in cancer treatment. Melanoma is the most common cancer in young adults ages 20-30, and it is the leading cause of cancer death in females ages 25-30. Non-modifiable risk factors include age, gender, and inherited predisposition to moles. As for modifiable risk factors, exposure to UV rays from the sun is well-established, but obesity has recently emerged as a factor through recent epidemiological and animal studies. Our results showed that obesity modulates the expression of the transcription factor Snai1, which has been shown to be a key gene in the regulation of the Epithelial-to-Mesenchymal Transition (EMT). Serum from obese ob/ob mice, as well as conditioned media from 3T3L1 adipocytes, increased the invasive ability of melanoma cells and the expression of the transcription factor Snai1. Yet, the cytokine IL-6 may not be a critical component of obesity-mediated B16BL6 melanoma cell invasiveness.Item The cAMP-mediated induction of tyrosinase activity in melanoma cell cultures by liposomes(Texas Tech University, 1983-12) Bayat-Makou, Christine L.We have examined the effects of liposome-encapsulated cAMP on tyrosinase activity in S91 Cloudman mouse melanoma cells. Existing methodology for liposome-formation and uptake by cells was adapted to our system. Different methods of liposome formation were examined and the parameters of total lipid concentration, cell number, liposome size, length of incubation time, and concentration of encapsulated cAMP were defined. Incubation of S91 Cloudman mouse melanoma cell cultures with large unilamellar liposomes containing 0,1 M cAMP resulted in maximeil incorporation of vesicle contents in 60 minutes. However, a 3 hour incubation of melanoma cells with liposome-encapsulated cA14P was needed for a maximal increase in tyrosinase activity (200% over control values), These results provide evidence that the effect of MSH on tyrosinase activity in mouse melanoma cells is mediated by cAMP, To investigate the possible role of cAMP dependent protein kinase in the action of MSH, the catalytic subunit of protein kinase was partially purified. Attempts of encapsulation of the activated enzyme were met with limited success.