Date of Award

Spring 5-16-2018

Degree Type

Thesis

Degree Name

Master of Science - Biotechnology

Department

Biology

First Advisor

Dr. Beatrice A. Clack

Second Advisor

Dr. Josephine Taylor

Third Advisor

Dr. Rebecca Parr

Fourth Advisor

Dr. Stephen Mullin

Abstract

Colorectal cancer is the third most common type of cancer in the world and the second leading cause of death among humans. Extracts of water soluble compounds from the roots and leaves of Rumex crispus were screened for compounds that induced apoptosis in DLD-1 cells. A compound referred to as L19 was isolated using Accelerated Solvent Extraction (ASE) followed by High Performance Liquid Chromatography (HPLC). Gas chromatography coupled with mass spectrometry was used to identify the L19 as a tetrahydrofuran with a molecular weight of 72.11 g/mole. Each HPLC fraction resulted in 94.4% purity. In the present research, specific genes involved in apoptosis induction with the treatment of the DLD-1 cells with L19 were identified. Apoptosis was measured by detecting levels of caspase 3 and 7 using the APO-one assay after treating synchronized DLD-1 cells with varying concentrations of L19 for 24 hrs. Furthermore, the mechanism of apoptosis was determined using quantitative real-time polymerase chain reaction (qRT-PCR) and gene specific primers for caspases 6, 8, 10 (extrinsic pathways), 9 (intrinsic), 1, 4, 5 and 12 (ER stress). RT2 Profiler PCR array for human apoptosis genes was used to explore additional changes in apoptotic gene expression. Microarray analysis was performed using the Human OneArray® Microarray from Phalanx Biotech Group to determine which genes of other key cellular pathways were affected by L19. Exposure of the DLD-1 cells to L19 for 6, 8, 12, and 24 hours and subsequent gene expression analysis indicated primarily that ER stress was the mechanism of apoptosis along with inflammation. The qRT-PCR results showed CASP 1 and 12 exhibited up-regulation by 12 hours, after which, results from the RT² Profiler™ PCR array showed BCL2 an inhibitor for apoptosis down-regulated. Apoptosis genes, CASP 3, APAF-1 and BAX and those involved in ER Stress, BH3, FAS, and FASLG were up-regulated as were the genes involved in inflammation, CASP 1, 12. Based on these results, L19 induces ER stress, AIF pathway (apoptosis pathways) and inflammatory pathways were shown. The microarray results show many more neurological, metabolic, and cell cycle processes affected by L19 that need to be investigated.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Sunday, May 19, 2019

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