Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Thesis - MUSC Only

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Azizul Haque

Second Advisor

Natalie Sutkowski

Third Advisor

Laura Kasman

Fourth Advisor

E. Bartee

Fifth Advisor

S. Eblen

Abstract

Diffuse large B-cell lymphoma (DLBCL), a subtype of non-Hodgkin’s lymphoma, is the most common lymphoid malignancy in the western world, accounting for almost 90% of all aggressive B-cell lymphoma cases. Treatment of DLBCL has been greatly improved in recent years with the addition of the monoclonal antibody Rituximab to the gold standard CHOP chemotherapy regimen, but these treatments are often ineffective in patients with highly aggressive disease or in patients of advanced age. These chemotherapeutics also present the issue of severe toxicity to the patient, further complicating the treatment of these patients. Thus, research has focused lately on natural products that can selectively kill malignant tumors while displaying a reduced host toxicity profile. Here, I investigated for the first time a natural extract from the Ganoderma lucidum mushroom, Ganoderic Acid DM (GA-DM), induces apoptosis in the murine B-cell lymphoma cell line, A20. My initial experiments using the MTS assay showed a reduction of enzymatic activity of A20 cells when treated with different concentrations of GA-DM after 24 hours of treatment with comparable results to treating the cells with Doxorubicin. These results suggested a reduction in cell viability which spurred further investigation into potential changes in the cell death pathway proteins as a result of treatment with GA-DM. MTS experiments also showed reduced viability of primary B cells when treated with GA-DM. Mechanistic studies performed through Western blot analysis confirmed the induction of caspase independent cell death in the murine DLBCL cell line A20 through increased PARP-l cleavage and AIF expression. Further investigation of changes in cell death and survival proteins through Western blot analysis added further support to the induction of caspase independent cell death in the A20 cells when treated with GA-DM. There was not an upregulation of the apoptotic protein Bax but there was a downregulation of the anti-apoptotic protein Bcl-2 and the survival protein Survivin. Western blot analysis of autophagic proteins showed a downregulation of Beclin-1 and LC3 thus ruling out autophagy as a process involved in the death of the A20 cells in regards to GA-DM treatment. TMRE mitochondrial stability assay confirmed these findings where GA-DM caused less depolarization in the A20 cell lines as determined by flow cytometry. Further study through flow cytometry using Annexin V and P1 staining did not show a greater level of apoptotic cells when the cells are treated with GA-DM which would be observed in the conventional apoptotic pathways. These findings support the potential use of GA-DM as a novel chemotherapeutic in the treatment of DLBCL and could improve the treatment of higher risk patients with advanced disease who cannot tolerate current chemotherapy treatment.

Rights

All rights reserved. Copyright is held by the author.

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