Graduation Year

2018

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Brant Burkhardt, Ph.D.

Committee Member

Meera Nanjundan, Ph.D.

Committee Member

Cecilia Nunes, Ph.D.

Committee Member

Stanley Stevens, Ph.D.

Keywords

Chemoresistance, Gemcitabine, PDAC, TCM

Abstract

Pancreatic adenocarcinoma (PDAC) remains one of the most lethal cancer types, with 92% of patients succumbing to the disease within five years. One of the hallmarks of this intractable disease is its nearly universal resistance to chemotherapeutic intervention. The deoxycytidine analog gemcitabine persists as the primary chemotherapy for advanced PDAC patients despite little improvement to its toxicity and tenuous efficacy over the past two decades. Plant-based drug discovery has a longstanding history and has led to some of the most significant drugs of the modern era, including some chemotherapeutic agents like paclitaxel and camptothecin. Our study analyzed the potential inhibitory effects of herbal extracts against pancreatic cancer cells. We selected a panel of extracts based on their history of pancreas-specific action and their current use in clinics which employ traditional Chinese medicine (TCM). Initial data indicated that the extract of Anemarrhena asphodeloides (AA) exhibited a significant dose-dependent decrease in PDAC cell viability. The potentially cytotoxic effects of AA had not previously been examined in pancreatic cancer prior to our study. We examined the effects of AA and one of its constituents, timosaponin-AIII (TAIII), as well as AA or TAIII plus gemcitabine on PDAC cell viability via MTT assay. Flow cytometric analysis revealed dysfunctional cell cycle progression in PDAC cells treated with AA and TAIII alone and in combination with gemcitabine. We utilized a magnetic bead-based multiplex assay to determine the effects of these treatments on the expression and activation of Akt signaling pathway members and observed a marked decrease in anti-apoptotic protein activation along with an increase in the activation of pro-apoptotic proteins compared to cells treated with gemcitabine alone. The effect of AA and TAIII on gemcitabine sensitivity in PDAC cells was elucidated through the determination of transcriptional and protein expression of gemcitabine uptake and metabolism mechanisms. Finally, a luminescent-based kinase assay revealed that activity of the rate-limiting kinase in gemcitabine activation, deoxycytidine kinase (dCK), was notably increased in the cells treated with AA or TAIII compared to gemcitabine. Taken together, the results of our present study implicates AA as a significant inhibitor of PDAC cell progression as well as a potential adjunctive therapeutic agent which can enhance gemcitabine efficacy within advanced pancreatic cancer.

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