Novel Dichloroacetate Prodrugs for Improved Targeting of Breast Cancer Chemotherapeutics
Abstract
It is well documented that most cancer cells utilize aerobic glycolysis or Warburg
metabolism instead of mitochondrial oxidative phosphorylation for cell energy production.
Dichloroacetate is a synthetic small molecule with proven anticancer effect. As a pyruvate
mimic, dichloroacetate is a glycolytic inhibitor that inactivates pyruvate dehydrogenase
kinase causing the activation of pyruvate dehydrogenase. This activity enables pyruvate
conversion to acetyl-CoA leading to mitochondrial remodeling via the reversal of the
Warburg effect and the release of pro-apoptotic mediators into the cytoplasm. The primary
aim of this dissertation is to synthesize dichloroacetate prodrugs that improve DCA targeting
to different breast cancer subtypes, triple negative and ER+ breast cancers to have enhanced
cancer cell death.
The general hypothesis and aims of this work are introduced in Chapter 1. A literature
review is provided on cancer etiology, breast cancer epidemiology, and the influence of DCA
on cancer, specifically breast cancer metabolism in Chapter 2.
Chapters 3 and 4 detail the physicochemical analysis of phenoxyethyl
dichloroacetamide (PE-DCA) and functionalized glucose-dichloroacetamide (G-DCA),
respectively. Physicochemical analysis include: LC/MS, FT-IR, SEM, P-XRD, and 13C and
1H NMR spectroscopy. Chapter 3 focuses specifically on the physicochemical analysis of
PE-DCA. PE-DCA was synthesized with a yield of 87.11%, and molecular weight was
quantified using LC/MS and corresponded to the expected molecular weight of 247 g/mol.
The chemical structure and formula of PE-DCA were confirmed using FTIR and NMR
spectroscopy. XRD and SEM analysis indicated that the morphological structure is different
suggesting that the structural integrity of native DCA in comparison to that of the native
DCA, which could influence cellular uptake of the prodrug. The physicochemical properties
of G-DCA are presented in Chapter 4. G-DCA was synthesized in a two-step process with
yields >97% for each step. Using LC/MS, the molecular weight was confirmed to be 502.09
g/mol. G-DCA’s chemical structure and formula were further confirmed using FTIR and
NMR spectroscopy. XRD and SEM analysis illustrate that G-DCA has a contrasting
morphology and composition than that of native DCA. The differences in the
physicochemical properties of the prodrugs can give insight on their metabolic profile.
The cell viability and toxicity activity of MDA-MB 231, MCF-7, and MCF-10A
(normal breast epithelial cells) when treated with PE-DCA and G-DCA for 24 h,
respectively, are described in Chapters 5 and 6. As compared to native DCA, with CC50
values of 25 mM and 25-30 mM in MDA-MB 231 and MCF-7 cell lines, respectively, CC50
values for PE-DCA (MDA-MB 231=14.72 µM; MCF-7=47.60 µM) and G-DCA (MDA-MB
231=16.22 µM; MCF-7=97.54 µM) were substantially lower, further proving that the
prodrugs enhance DCA’s targeting to cancer cells to reverse the Warburg effect.
The metabolic profiles of the two cancerous cell lines after treatment with G-DCA,
PE-DCA, and DCA in different media environments (pyruvate, low glucose and high
glucose) are elucidated in Chapter 7. Intracellular ROS and autophagosome production were
quantified in the various conditions to explore the relationship between ROS production and
the induction of autophagy when exposed to different media environments. Results for both
cancer cell lines indicate an increase intracellular ROS production in high glucose media
which further exploits the similarities in the metabolic profiles of breast cancer subtypes.
Moreover, related to autophagosome production, upon drug treatment in varying
concentrations and media conditions, the relationship between ROS and autophagy induced
apoptosis was explored.
The intracellular DCA uptake in MCF-10A, MCF-7, and MDA-MB 231 cell lines
after treatment with DCA, PE-DCA, and G-DCA are quantified in Chapter 8. Results showed
that the DCA prodrugs are more cell targeted to cancer cells than normal breast epithelial
tissues further proving that because of DCA conjugation, DCA’s target specificity is more
refined.
This dissertation gives a comprehensive analysis of the metabolic and
physicochemical characteristics of the novel DCA prodrugs and their influence on breast
cancer proliferation and cell death.
Table of Contents
Introduction -- Literature review -- Synthesis and physicochemical analysis of phenoxyethyl dichloroacetamide (PE-DCA) prodrug -- Physicochemical analysis of glucose dichloroacetamide (G-DCA) prodrug -- Biological analysis of breast cancer cell response to phenoxyethyl dichloroacetamide (PE-DCA) prodrug -- Biological analysis of breast cancer cell response to Glucose dichloroacetamide (G-DCA) prodrug -- Quantifying metabolic response in breast cancer cells after DCA prodrug treatment -- Quantifying intracellar uptake of dichloroacetate via LC-MS/MS -- Summary & future perspectives -- Appendix. Abbreviations
Degree
Ph.D. (Doctor of Philosophy)