Search

The primary aim of this project was to develop total syntheses of natural products kamalachalcone A and rottlerin, as their syntheses have never been reported. These two targets are structurally related, and thus the complete syntheses of these molecules would help explain their biosynthesis and key bond formations in nature. Novel chromene analogues with fused ring systems were synthesized by developing a method to generate ortho-quinone methide precursors, which could be reacted with various electron-rich alkenes to form fused heterocyclic rings. The results showed that electron-rich alkenes such as vinyl ether and styrene derivatives favour the cycloaddition reaction whereas electron poor alkenes do not undergo this reaction. The results also showed that electron-rich arenes could also furnish biarylmethane compounds, a potential strategy towards the synthesis of rottlerin. To explore the properties of ortho-quinone methides further, novel fluorinated dependensin analogues were synthesized by the FeCl3-catalyzed dimerization reaction of the corresponding fluorinated flavenes. The results suggested that the dimerization reaction of flavenes is controlled electronically, with electronegative fluorine substituents appearing to deactivate the overall nucelophilicity of flavenes, thus leading to lower yields of the dimeric products. Dimerization reactions were only successful with flavene scaffolds containing two or fewer fluorine substituents. By understanding the properties of ortho-quinone methides, kamalachalcone A was synthesized for the first time via the acid-catalyzed dimerization of red compound. The use of FeCl3 offered the best yield with short reaction time. The structurally similar compound, rottlerin, was synthesized by reacting 5-hydroxychromene with Eschenmoser’s salt to provide N,N-dimethylaminomethyl chromene as an ortho-quinone methide precursor, which upon heating with acetophenone, provided rottlerin. By understanding the formation of the side product rottlerone during the deprotection of MOM-protected chromene, the overall synthesis was re-designed and a new five-step gram scale synthesis of rottlerin was developed. Upon keeping rottlerin in DMSO, the chalcone moiety of rottlerin underwent cyclization leading to the formation of another natural product called isorottlerin, and this is the first report of the ability of rottlerin to undergo such a change.

Thyroid cancer is the most common endocrine cancer and its incidence rates continue to rise. Papillary thyroid cancer (PTC) is the most common type and it is generally treatable with surgery followed by radioactive iodine (RAI) therapy. Thyrotropin (TSH)-stimulated, Sodium/Iodide Symporter (NIS)-mediated RAI uptake (RAIU) is the basis for RAI therapy. While most patients show good prognosis, a subset of patients with advanced thyroid cancer do not benefit from RAI therapy due to reduced NIS expression/function. The objective of this study was to investigate the molecular mechanisms of NIS modulation and to identify novel reagents that selectively increase RAIU in thyroid cells, such that the subset of patients could benefit from RAI therapy. Most common genetic alterations in PTCs are the BRAF(V600E) mutation that activates MEK/ERK signaling and RET/PTC rearrangement that activates both PI3K/Akt/mTOR and Ras/BRAF/MEK/ERK pathways. Hsp90 is a chaperone to many of these oncoproteins. TGF-beta is a cytokine in invasive fronts of thyroid tumors, which also activates MEK/ERK. These signaling nodes not only contribute to tumor progression but also result in reduced NIS expression/function. Here we report that among cell culture-grade small molecule inhibitors tested, Akt inhibitor (Akti1/2) increased TSH-stimulated RAIU to the greatest extent in PCCl3 rat thyroid cells, when compared to inhibitors of MEK, PI3K or Hsp90. We identified Apigenin, a plant-derived flavonoid that further increased RAIU by increasing iodide influx rate in combination with Akti1/2 and this required p38 MAPK activity, but not PKC-delta. The effect was confirmed in BRAF(V600E) or RET/PTC1-expressing PCCl3 cells, and in primary thyroid tumor cells from TR-beta(PV/PV) mice.We then took advantage of small molecule inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF used in clinical trials for chemotherapy of solid tumors. As their toxicity profiles are well-accepted, if they increase thyroidal RAIU, they could be immediately employed in clinical trials to improve RAI treatment efficacy. We identified that PI3K inhibitor GDC-0941 outperformed other inhibitors in increasing TSH-stimulated RAIU in PCCl3 and BRAF(V600E)-expressing PCCl3 cells, by greatly decreasing iodide efflux rate. Alarmingly, TGF-beta dramatically reduced RAIU even upon treatment with inhibitors. Apigenin counteracted the RAIU reduction by TGF-beta by increasing NIS protein. Taken together, co-treatment of GDC-0941 with Apigenin may increase therapeutic efficacy of RAI in invasive fronts of thyroid cancer. While much is known about signaling nodes, little is known about microRNAs (miRs) regulating NIS and RAIU. We identified that miR-339-5p decreased endogenous NIS mRNA and RAIU in MCF-7 human breast cancer cells and in PCCl3 cells. Among 38 miRs deregulated by TGF-beta, Akti-1/2 or 17-AAG in PCCl3 cells, 18 were conserved in humans. One of the 18 miRs, miR-195, was predicted to bind to hNIS-3’UTR and its overexpression decreased hNIS-mediated RAIU in MCF-7 cells. MiR-339-5p was modestly increased, yet miR-195 was significantly decreased in PTCs. Expression profiles of the 18 miRs were able to separate most PTCs from non-malignant thyroid tissues. Taken together, we hope that these studies would serve as valuable tools to develop novel strategies for optimal use of RAI in clinical management of thyroid cancer.

Mammalian intracellular protein homeostasis is maintained by a process called macroautophagy, or autophagy, and is a major protein degradation mechanism induced by cellular stress such as nutrient starvation. The discovery of key autophagy genes as well as signaling pathways involved in the regulation of autophagy has elucidated the essential protein machinery involved in autophagosome formation, fusion, and degradation. However, the specific mechanism as to how these genes and kinase pathways regulate autophagy is largely unknown. In addition to normal cellular physiology, autophagy has been implicated in the pathophysiology of cancer. This is evidenced by an increase in the frequency of spontaneous malignancies by heterozygous disruption of the autophagy gene Beclin-1; suggesting that Beclin-1 functions as a tumor suppressor gene. We show that Beclin-1 co-immunoprecipitates with the Class III PI3K hVps34 and its myristoylated protein adapter p150, and that this interaction requires amino acids 80-107 of Beclin-1. We also demonstrate that upon autophagic stimulation, the Beclin-1-hVps34-p150 complex is recruited to the membrane fraction in MCF-7 cells.Rottlerin is a cytotoxic compound reported to increase the presence of autophagic vacuoles in the cytoplasm of pancreatic cells, membrane association of GFP-tagged LC3 to autophagosomes, and a marked induction of LC3-II protein, all important hallmarks of autophagy. Additionally, inhibition of Beclin-1 expression by RNAi was shown to inhibit Rottlerin-induced autophagy. Rottlerin was initially reported to specifically inhibit Protein Kinase C delta (PKCδ), and has also been shown to uncouple mitochondrial oxidative phosphorylation. The second portion of the dissertation aimed to explore the hypothesis that the cytotoxic effects of Rottlerin are related to its regulation of autophagy. Our results demonstrate that Rottlerin causes an accumulation of the autophagosomal marker LC3-II in MCF-7 and ZR75 breast cancer cells. This increase in accumulation of LC3-II was caused mainly by an impairment of the autophagy pathway at the lysosomal degradation step. However, a modest increase in autophagosome biogenesis contributes to the accumulation of LC3-II. Our studies concluded that exposure of breast cancer cells to Rottlerin induces caspase-independent cell death by reducing mitochondrial membrane potential (MMP), stimulating ER stress, and inhibiting autophagosome fusion with lysosomes, thereby rendering the autophagy survival pathway ineffective.