Engineered bacterial membrane pore for drug delivery to human cancer cells : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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College Station, Tex. :
Texas A&M University System Health Science Center,
2008.
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| Subjects: |
| Abstract: | ABSTRACT: [alpha]-Hemolysin ([alpha]HL) secreted from Staphylococcus aureus, as a water soluble monomeric protein, is a [beta] barrel pore-forming toxin. [alpha]HL binds to the membrane of a susceptible cell and lyses cells. The sensitivity to [alpha]HL varies by different cell types. The sensitvity of rabbit erythrocytes to [alpha]HL is, for example. 1000-fold higher than human erythrocytes. An engineered alpha-hemolysin, [alpha]HL chimera with galectin-1 ([alpha]HLG1), was constructed by fusing galection-1 to the C-terminus of [alpha]HL. Like vibrio cholerae cytolysin, [alpha]HLG1 has ben designed to have a cytolytic domain, a linker, and a lectin domain. In the present study, the hemolytic activity of [alpha]HLG1 toward human erythrocytes was tested compared with that of [alpha]HL. And the hemolytic acitivity of [alpha]HLG1 has about 150-fold higher than that of [alpha]HL toward human erythrocytes. These results suggest that the receptor binding of the lectin domain of [alpha]HLG1 contributes to the activities including the binding and the hemolysis on hRBC. And it has been discovered that they exist on human erythrocytes membrane. In addition, human promyelocytes (HL-60) are more sensitive to [alpha]HLG1 than [alpha]HL. The lytic activity of [alpha]HLG1 toward HL-60 is at least 2-fold higher that that of [alpha]HL. Based on this result, we can conclude that the specific receptor of the HL-60 cell membranes binding of the lectin domain (galectin-1 domain) raises the activity of [alpha]HLG1 toward HL-60 cells compared to that of [alpha]HL. Like, the activities of [alpha]HLG1 toward HT-1080 cells, human fibrosarcomas, are also higher than those of [alpha]HL. To further increase the specificity of [alpha]HLG1 toward the target cells, a protease-activable mutant of [alpha]HLG1 (PAM[alpha]HLG1 was constructed, by using the mutant containing a nick between position 131 and 132, which divided [alpha]HLG1 into two domains (N- and C- terminal domains of PAM[alpha]HLG1) and introducing the 13 amino acids containing specific protease cleavage site at position 132 of the C- terminal domain of PAM[alpha]HLG1. PAM[alpha]HLG1 is fully active toward HT-1080 cells containing tumor proteases to specifically activate PAM[alpha]HLG1. Interestingly, PAM[alpha]HL is inactive with HL-60 cells and erythrocytes. These results suggest that the activity of specific protease is one of the substantial magnitudes for a cell targeting specificity. In a preliminary study , pores formed by [alpha]HLG1/PAM[alpha]HLG1 were tested to determine if they were suitable for drug delivery by using methotrexate-Alexa 488 conjugate (MTX-Alexa). Remarkably, the cellular content of MTX-Alexa 488 is membrane-permeable itself. In summary, engineered alpha-hemolysin for a cancer cell-targeting toxin with two-fold specificity provides a valuable approach in the cancer therapy. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy May 2008." Approved as to style and content by: Hagan Bayley, Nick Pace, Rajesh Miranda, Patraicia LiWang, J. Martin Scholtz. |
| Physical Description: | x, 104 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 99-103). |