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theses

ETD doctoral

Cancer progression into metastasis is an incredibly complex, multistep process that is the major cause of cancer-related deaths. Although new potentially therapeutic metallodrugs are being synthesized at high rates, there is currently no robust method for evaluation of toxicity or efficacy in mice or other model organisms. In fact, New Anti-Tumor Metastasis Inhibitor (NAMI-A), a ruthenium (Ru)-based complex, showed excellent anti-metastatic properties in vitro and in the nude mouse model (the current model systems), but failed clinical trials. As such, there is a substantial need for an alternative method of evaluating therapeutic efficacy, to prioritize the most promising of candidate compounds.

In order to evaluate zebrafish as a potential model for metallodrug evaluation, cisplatin, a widely used platinum (Pt)-based chemotherapeutic drug, was utilized as a proof-of-principle compound. We coupled a modified OECD FET (Organisation for Economic Cooperation and Development Fish Embry Acute Toxicity) protocol with lesion identification, morphological endpoints and metallodrug up-take quantification by Inductively Coupled Plasma Mass Spectrometry (ICPMS). By coupling these experiments with ICPMS analysis of waterborne solutions and larval tissue we were able to determine drug uptake associated with dose-dependent endpoints with cisplatin as well as two novel ruthenium-based metallodrugs: PMC79 and LCR134. This provided a method to overcome a common limitation of the zebrafish model: waterborne dose delivery and identify doses for future experiments. Lowest observed adverse effect levels (LOAELs): 3.75 mg/L, 3.1 mg/L and 17.4 mg/L for cisplatin, PMC79, and LCR134, respectively.

These compounds were then evaluated for their anti-proliferation and angiogenic capabilities. Gene expression of Vascular Endothelial Growth Factors (vegfa and c), Wingless/Integrated signaling (wnt 3a and 8a) and Hypoxia-inducible factor 1-alpha (hif1-α) were evaluated. Significant inhibition of mRNA expression of all genes with the exception of wnt3a was associated with PMC79 metallodrug exposure. However, LCR134 exposure did not cause significant changes in gene expression. In addition to PMC79 exposure significantly decreasing gene expression, the sub-intestinal blood vessels showed significantly less branching which indicates inhibition of angiogenesis. LCR134 did deviate from control branching. Lastly, PMC79, LCR134 and cisplatin were utilized in a tail fin regeneration assay conducted concurrently with whole mount immunofluorescence using proliferating cell nuclear antigen (PCNA), a marker of proliferation. Our findings demonstrated similar inhibition profiles with cisplatin. This was a marked finding as cisplatin was used at levels with a higher propensity for toxicity than either Ru metallodrugs.

Furthermore, we were able to recapitulate the in vitro mechanisms of LCR134 and PMC79 in the teleost model. These metallodrugs were previously investigated in several cancer cells lines; LCR134 was found to significantly inhibit P-glycoprotein (Pgp) efflux pump also known as multidrug resistance protein 1; mdr1. This protein is often found to be correlated with increased drug resistance. PMC79 has been found to initiate an apoptotic cascade through disrupting cytoskeleton (F-actin) in the plasma membrane. Gene expression of b-actin after PMC79 exposure was significantly down-regulated; however, gene expression of mdr1 after LCR134 was not affected. In vivo Pgp inhibition showed significant retention of the Pgp-specific fluorescent substrate Rhodamine 123 (Rh123) after treatment with LCR134 and a clinically-relevant inhibitor, cyclosporine A. PMC79 in vivo assessment of membrane cytoskeletal impact demonstrated a significant decrease in the presence of cytoskeletal structures. These experiments provided insight to the retention of mechanistic activity in whole organism models.

The objective of this project was to assess the specific modes of action for novel metallodrugs in a higher through-put, alternative animal model. The culmination of this work has shown the zebrafish model as a powerful platform for the evaluation of novel Ru metallodrugs as well as their promising candidacy for evaluation in higher organisms.

ETD_11007

Ph.D.

Includes bibliographical references

doi:10.7282/t3-ajmb-1006

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